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The maternal-to-zygotic transition (MZT) is a conserved embryonic process in animals where developmental control shifts from the maternal to zygotic genome. A key step in this transition is zygotic transcription, and deciphering the MZT requires classifying newly transcribed genes. However, due to current technological limitations, this starting point remains a challenge for studying many species. Here we present an alternative approach that characterizes transcriptome changes based solely on RNA-seq data. By combining intron-mapping reads and transcript-level quantification, we characterized transcriptome dynamics during the Drosophila melanogaster MZT. Our approach provides an accessible platform to investigate transcriptome dynamics that can be applied to the MZT in non-model organisms. In addition to classifying zygotically transcribed genes, our analysis revealed that over 300 genes express different maternal and zygotic transcript isoforms due to alternative splicing, polyadenylation, and promoter usage. The vast majority of these zygotic isoforms have the potential to be subject to different regulatory control, and over two-thirds encode different proteins. Thus, our analysis reveals an additional layer of regulation during the MZT, where new zygotic transcripts can generate additional proteome diversity.

Purpose: N6-methyladenosine (m6A) methylation was the most abundant internal modification on messenger RNAs in eukaryotes. This study intended to explore the role of m6A methylation in endometrial cancer (EC). Materials and Methods: The m6A-sequencing data "GSE93911" of human EC were downloaded from Gene Expression Omnibus database. Hisat2 software and MACS2 were used to perform the alignment of reads and m6A methylation peak calling, and the peaks were annotated using Chipseeker. Then, differential m6A methylation peaks between normal and tumor samples were analyzed, followed by the functional enrichment analysis of the differentially methylated genes in promoter and 3' untranslated region (UTR) using Clusterprofiler. Based on the 450K methylated chip data, gene expression and clinical data in The Cancer Genome Atlas, the differentially methylated genes were verified, followed by Cox univariate/multivariate regression analysis and survival analysis. Finally, a risk prognosis model was constructed. Results: The m6A peak number was decreased in EC. The distribution of m6A peaks was highly enriched near transcriptional start site, in promoter, UTR, intron and exon, followed by distal intergenic. A total of 581 differentially methylated genes (361 hyper- and 220 hypomethylated genes) were identified in promoter and UTR regions that were enriched in insulin resistance (IR) and extracellular matrix (ECM). A total of 181 genes with significant differential expressions and differential methylation site in EC were selected. Of which, 31 genes were correlated with survival, and an 11-gene risk prognosis model was identified, including GDF7, BNC2, SLC8A1, B4GALNT3, DHCR24, ESRP1, HOXB9, IGSF9, KIAA1324, MSnX1, and PHGDH. Conclusion: The m6A methylation regulated EC progression by targeting the genes related to IR and ECM. A 11-gene risk prognosis model was identified to predict survival of patients with EC.

DNA double-strand breaks are among the most toxic lesions that can occur in a genome and their faithful repair is thus of great importance. Recent findings have uncovered local transcription that initiates at the break and forms a non-coding transcript, called damage-induced long non-coding RNA (dilncRNA), which helps to coordinate the DNA transactions necessary for repair. We provide nascent RNA sequencing-based evidence that RNA polymerase II transcribes the dilncRNA in Drosophila and that this is more efficient for DNA breaks in an intron-containing gene, consistent with the higher damage-induced siRNA levels downstream of an intron. The spliceosome thus stimulates recruitment of RNA polymerase II to the break, rather than merely promoting the annealing of sense and antisense RNA to form the siRNA precursor. In contrast, RNA polymerase III nascent RNA libraries did not contain reads corresponding to the cleaved loci and selective inhibition of RNA polymerase III did not reduce the yield of damage-induced siRNAs. Finally, the damage-induced siRNA density was unchanged downstream of a T8 sequence, which terminates RNA polymerase III transcription. We thus found no evidence for a participation of RNA polymerase III in dilncRNA transcription in cultured Drosophila cells.

Methylmalonic acidemia (MMA) is a metabolic disorder most commonly caused by mutations in the methylmalonyl-CoA mutase (MMUT) gene. Although adeno-associated viral (AAV) gene therapy has been effective at correcting the disease phenotype in MMA mouse models, clinical translation may be impaired by loss of episomal transgene expression and magnified by the need to treat patients early in life. To achieve permanent correction, we developed a dual AAV strategy to express a codon-optimized MMUT transgene from Alb and tested various CRISPR-Cas9 genome-editing vectors in newly developed knockin mouse models of MMA. For one target site in intron 1 of Alb, we designed rescue cassettes expressing MMUT behind a 2A-peptide or an internal ribosomal entry site sequence. A second guide RNA targeted the initiator codon, and the donor cassette encompassed the proximal albumin promoter in the 5′ homology arm. Although all editing approaches were therapeutic, targeting the start codon of albumin allowed the use of a donor cassette that also functioned as an episome and after homologous recombination, even without the expression of Cas9, as an integrant. Targeting the albumin locus using these strategies would be effective for other metabolic disorders where early treatment and permanent long-term correction are needed., Graphical Abstract, Methylmalonic acidemia, MMUT subtype, is a severe metabolic disorder treated by elective liver transplantation. We developed several therapeutic genome-editing approaches, with and without nucleases, to insert the MMUT gene into the 5′ end of the albumin locus using AAV vectors. Our results should be broadly extendable other metabolic disorders.

GGGGCC (G4C2) repeat expansion in the first intron of C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-containing RNA is translated into dipeptide repeat (DPR) proteins, some of which are neurotoxic. Using dynamic ribosome profiling, we identified three translation initiation sites in the intron upstream of (G4C2) repeats; these sites are detected irrespective of the presence or absence of the repeats. During translocation, ribosomes appear to be stalled on the repeats. An AUG in the preceding C9ORF72 exon initiates a uORF that inhibits downstream translation. Polysome isolation indicates that unspliced (G4C2) repeat-containing RNA is a substrate for DPR protein synthesis. (G4C2) repeat-containing RNA translation is 5′ cap-independent but inhibited by the initiation factor DAP5, suggesting an interplay with uORF function. These results define novel translational mechanisms of expanded (G4C2) repeat-containing RNA in disease.

This study investigated the effects of single nucleotide polymorphisms (SNPs) of the VEGF （vascular endothelial growth factor） gene, which are associated with susceptibility to age-related macular degeneration (AMD), on the expression of VEGF proteins (VEGF165 and VEGF165b) and their role in cell proliferation and apoptosis in human retinal vascular endothelial cells (hRVECs). Cell viability and VEGF165 and VEGF165b expressions were evaluated in hRVECs transfected with VEGF genes containing different SNPs (rs3025039, rs3025033, and rs10434). The Cell Counting Kit 8 assay, quantitative real-time PCR, western blotting, TUNEL assay, and enzyme-linked immunosorbent assay were used to examine the effects of VEGF gene SNPs on cell viability, VEGF165 and VEGF165b expressions, and cell apoptosis in hRVECs. The interaction and localization of the RNA-binding protein alternative splicing factor/splicing factor 2 (ASF/SF2) were assessed using RNA pull-down. Although VEGF165 expression decreased, VEGF165b levels increased significantly in hRVECs transfected with rs3025039, which decreased cell viability and induced apoptosis. The SNPs rs3025033 and rs10434 had no significant effects on VEGF165b protein production and apoptosis; however, they promoted cell proliferation. SNPs affected the interaction between RNA and ASF/SF2, a splicing factor for intron retention. Insulin-like growth factor-1 treatment induced the expression of VEGF165, but not VEGF165b, whereas SRPIN340 treatment, an inhibitor of ASF/SF2, increased VEGF165b protein levels. VEGF gene sequence variations affected hRVEC proliferation and apoptosis via alternative gene splicing. Thus, the regulation of splicing via ASF/SF2 could be a potential strategy in treating pathological neovascularization in patients with AMD.

Hemocyanin is the oxygen transport protein of most molluscs and represents an important physiological factor that has to be well-adapted to their environments because of the strong influences of abiotic factors on its oxygen affinity. Multiple independent gene duplications and intron gains have been reported for hemocyanin genes of Tectipleura (Heterobranchia) and the caenogastropod species Pomacea canaliculata, which contrast with the uniform gene architectures of hemocyanins in Vetigastropoda. The goal of this study was to analyze hemocyanin gene evolution within the diverse group of Caenogastropoda in more detail. Our findings reveal multiple gene duplications and intron gains and imply that these represent general features of Apogastropoda hemocyanins. Whereas hemocyanin exon–intron structures are identical within different Tectipleura lineages, they differ strongly within Caenogastropoda among phylogenetic groups as well as between paralogous hemocyanin genes of the same species. Thus, intron accumulation took place more gradually within Caenogastropoda but finally led to a similar consequence, namely, a multitude of introns. Since both phenomena occurred independently within Heterobranchia and Caenogastropoda, the results support the hypothesis that introns may contribute to adaptive radiation by offering new opportunities for genetic variability (multiple paralogs that may evolve differently) and regulation (multiple introns). Our study indicates that adaptation of hemocyanin genes may be one of several factors that contributed to the evolution of the large diversity of Apogastropoda. While questions remain, this hypothesis is presented as a starting point for the further study of hemocyanin genes and possible correlations between hemocyanin diversity and adaptive radiation. Supplementary Information The online version contains supplementary material available at 10.1007/s00239-021-10036-y.

Marek's disease (MD), an immunosuppressive disease induced by Marek's disease virus (MDV), provides an ideal model for studying diseases caused by a carcinogenic virus. CD79B is a B-cell antigen receptor complex-associated protein β-chain precursor which is involved in the activation, proliferation, differentiation of B-cell and the transmission of downstream signals. This study analyzed CD79B gene mRNA expression and methylation by two schemes #20 (5´ flanking to intron 1) and #27 (intron 2 to intron 3), between MDV-infected tumorous spleens (TS) and non-infected spleens (NS). Results showed that average methylation levels of CpGs in #20 and #27 were higher in TS than in NS (P

Loss of the awn in some cereals, including sorghum, is a key transition during cereal domestication or improvement that has facilitated grain harvest and storage. The genetic basis of awn loss in sorghum during domestication or improvement remains unknown. Here, we identified the awn1 gene encoding a transcription factor with the ALOG domain that is responsible for awn loss during sorghum domestication or improvement. awn1 arose from a gene duplication on chromosome 10 that translocated to chromosome 3, recruiting a new promoter from the neighboring intergenic region filled with “noncoding DNA” and recreating the first exon and intron. awn1 acquired high expression after duplication and represses the elongation of awns in domesticated sorghum. Comparative mapping revealed high collinearity at the awn1 paralog locus on chromosome 10 across cereals, and awn growth and development were successfully reactivated on the rice spikelet by inactivating the rice awn1 ortholog. RNA-seq and DAP-seq revealed that as a transcriptional repressor, AWN1 bound directly to a motif in the regulatory regions of three MADS genes related to flower development and two genes, DL and LKS2, involved in awn development. AWN1 downregulates the expression of these genes, thereby repressing awn elongation. The preexistence of regulatory elements in the neighboring intergenic region of awn1 before domestication implicates that noncoding DNA may serve as a treasure trove for evolution during sorghum adaptation to a changing world. Taken together, our results suggest that gene duplication can rapidly drive the evolution of gene regulatory networks in plants.

Alu RNA are implicated in the poor prognosis of several human disease states. These RNA are transcription products of primate specific transposable elements called Alu elements. These elements are extremely abundant, comprising over 10% of the human genome, and 100 to 1000 cytoplasmic copies of Alu RNA per cell. Alu RNA do not have a single universal functional role aside from selfish self-propagation. Despite this, Alu RNA have been found to operate in a diverse set of translational and transcriptional mechanisms. This review will focus on the current knowledge of Alu RNA involved in human disease states and known mechanisms of action. Examples of Alu RNA that are transcribed in a variety of contexts such as introns, mature mRNA, and non-coding transcripts will be discussed. Past and present challenges in studying Alu RNA, and the future directions of Alu RNA in basic and clinical research will also be examined.

© 2022 Prudêncio et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society. This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/., Widespread cotranscriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure cotranscriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nt of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes, Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravel novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo., This work was supported by funding to M.C.-F. (Fundação para a Ciência e Tecnologia, FCT/Ministério da Ciência, Tecnologia e Ensino Superior - Fundos do Orçamento de Estado [UIDB/50005/2020], and FCT/ FEDER/POR Lisboa 2020, Programa Operacional Regional de Lisboa PORTUGAL 2020, grant LISBOA-01-0145-FEDER-016394) and to R.G.M (FCT grant PTDC/BIA-BID/28441/2017). P.P. was a recipient of an FCT fellowship (SFRH/BD/109689/2015). R.S. was a recipient of an EMBO Long-Term Fellowship (EMBO ALTF 101-2019). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreements no. 842695 (Marie Skłodowska- Curie Actions) and no. 857119 (RiboMed)

Background Abnormal alternative splicing is frequently associated with carcinogenesis. In B-cell acute lymphoblastic leukemia (B-ALL), double homeobox 4 fused with immunoglobulin heavy chain (DUX4/IGH) can lead to the aberrant production of E-26 transformation-specific family related gene abnormal transcript (ERGalt ) and other splicing variants. However, the molecular mechanism underpinning this process remains elusive. Here, we aimed to know how DUX4/IGH triggers abnormal splicing in leukemia. Methods The differential intron retention analysis was conducted to identify novel DUX4/IGH-driven splicing in B-ALL patients. X-ray crystallography, small angle X-ray scattering (SAXS), and analytical ultracentrifugation were used to investigate how DUX4/IGH recognize double DUX4 responsive element (DRE)-DRE sites. The ERGalt biogenesis and B-cell differentiation assays were performed to characterize the DUX4/IGH crosslinking activity. To check whether recombination-activating gene 1/2 (RAG1/2) was required for DUX4/IGH-driven splicing, the proximity ligation assay, co-immunoprecipitation, mammalian two hybrid characterizations, in vitro RAG1/2 cleavage, and shRNA knock-down assays were performed. Results We reported previously unrecognized intron retention events in C-type lectin domain family 12, member A abnormal transcript (CLEC12Aalt ) and chromosome 6 open reading frame 89 abnormal transcript (C6orf89alt ), where also harbored repetitive DRE-DRE sites. Supportively, X-ray crystallography and SAXS characterization revealed that DUX4 homeobox domain (HD)1-HD2 might dimerize into a dumbbell-shape trans configuration to crosslink two adjacent DRE sites. Impaired DUX4/IGH-mediated crosslinking abolishes ERGalt , CLEC12Aalt , and C6orf89alt biogenesis, resulting in marked alleviation of its inhibitory effect on B-cell differentiation. Furthermore, we also observed a rare RAG1/2-mediated recombination signal sequence-like DNA edition in DUX4/IGH target genes. Supportively, shRNA knock-down of RAG1/2 in leukemic Reh cells consistently impaired the biogenesis of ERGalt , CLEC12Aalt , and C6orf89alt . Conclusions All these results suggest that DUX4/IGH-driven DNA crosslinking is required for RAG1/2 recruitment onto the double tandem DRE-DRE sites, catalyzing V(D)J-like recombination and oncogenic splicing in acute lymphoblastic leukemia.

Background. Ischemic stroke (IS) is the most common form of cerebrovascular accident which its precise etiology remains mysterious. Renalase is a catecholamine-degrading enzyme playing a major role in blood pressure control. Recent studies show the effect of renalase activity on various diseases like IS. In the current study, we examined the possible effects of renalase gene (RNLS) rs2576178 and rs10887800 variants at the 5 ′ -flanking and intron 6 regions on IS, respectively. Methods. One hundred and fifty-four IS patients younger than 50 years and 165 age- and sex-matched controls were recruited in the study. For genotyping of rs2576178 and rs10887800 variants, the PCR-RFLP method was used. Results. The RNLS rs10887800 AG genotype was more repeated in IS patients, but the difference was marginally nonsignificant ( P = 0.054 ). This variant was associated with IS in the overdominant model, and the AG genotype is associated with a1.6-fold increased risk of IS compared to AA+ GG genotypes ( OR = 1.6 , 95% CI: 1-2.5, P = 0.033 ). No relationship was observed between RNLS rs2576178 polymorphism and IS in all genetic models. The findings of the haplotype and combination effects of rs10887800 and rs2576178 variants on IS showed no significant association. The in silico analysis showed no effect of rs2576178 and rs10887800 polymorphisms in the RNA structure, but the alteration of RNA sequence in rs2576178 results in the lack of a MBNL1 protein binding site. Conclusions. RNLS rs10887800 but not rs2576178 polymorphism was associated with IS susceptibility in the overdominant model (AG vs AA+ GG genotypes).

TP53 is one of the most ubiquitously altered genes in human cancer. The biological impact of rare variants, particularly those located within noncoding regions, remains poorly understood. From interrogation of clinical massively parallel sequencing data from over 55,000 tumors, which included 23,330 tumors with known TP53 mutations, TP53 intron 4 nucleotide substitutions at position c.375+5G were identified in 45 tumors (0.2% of TP53-mutated cancers), comprising cancers of different organ sites. Loss-of-heterozygosity or a second-hit somatic TP53 mutation was observed in 34 of 40 (85%) informative cases. RT-PCR analysis showed the c.375+5G>T variant to be associated with aberrantly spliced TP53 mRNA transcripts with concomitant loss of the normal transcript. Immunohistochemical staining for p53 was performed on a representative subset of tumors with TP53 c.375+5G variants (n = 14), all of which showed loss of protein expression (100%; n = 13 complete loss, n = 1 subclonal loss). Our data are consistent with classification of TP53 c.375+5G variants as deleterious intronic mutations that interfere with proper mRNA splicing, ultimately resulting in loss of expression of functional p53 protein. The clinical scenario of a tumor with loss of p53 immunohistochemical staining, yet lacking a detectable TP53 exonic mutation, should therefore prompt consideration of splice-altering intronic variants.

Trehalose plays an important role in plant metabolism, growth development, and stress tolerance. Trehalose-6-phosphate synthase gene (TPS) and trehalose-6-phosphate phosphatase gene (TPP) are vital for the synthesis of trehalose. Populus is a prominent perennial woody plant, in which systematic genome-wide analysis of the TPS and TPP family is limited. In this study, 13 PtTPS and 10 PtTPP genes were identified in the Populus genome. Phylogenetic analysis indicated PtTPS and PtTPP genes were both divided into two subfamilies, and gene members of each subfamily have highly conserved intron structures. Analysis of cis-acting elements showed that PtTPS and PtTPP genes were involved in plant hormones and environmental stress responses. Expression profiles also found PtTPSs and PtTPPs expressed differently in response to salt stress, cold, mechanical damage, salicylic acid, and methyl jasmonate treatment. Furthermore, reverse transcription quantitative real-time PCR results found PtTPSs and PtTPPs displayed a specific expression pattern in the seven developmental stages of Populus male and female floral buds. This work will not only lead a foundation on reveal the functions of PtTPS and PtTPP gene families in trehalose regulation of poplar but also provide references to related trehalose research in other perennial plants.

Cleavage factor polyribonucleotide kinase subunit 1 (CLP1), an RNA kinase, plays essential roles in protein complexes involved in the 3'-end formation and polyadenylation of mRNA and the tRNA splicing endonuclease complex, which is involved in precursor tRNA splicing. The mutation R140H in human CLP1 causes pontocerebellar hypoplasia type 10 (PCH10), which is characterized by microcephaly and axonal peripheral neuropathy. Previously, we reported that RNA fragments derived from isoleucine pre-tRNA introns (Ile-introns) accumulate in fibroblasts of patients with PCH10. Therefore, it has been suggested that this intronic RNA fragment accumulation may trigger PCH10 onset. However, the molecular mechanism underlying PCH10 pathogenesis remains elusive. Thus, we generated knock-in mutant mice that harbored a CLP1 mutation consistent with R140H. As expected, these mice showed progressive loss of the upper motor neurons, resulting in impaired locomotor activity, although the phenotype was milder than that of the human variant. Mechanistically, we found that the R140H mutation causes intracellular accumulation of Ile-introns derived from isoleucine pre-tRNAs and 5' tRNA fragments derived from tyrosine pre-tRNAs, suggesting that these two types of RNA fragments were cooperatively or independently involved in the onset and progression of the disease. Taken together, the CLP1-R140H mouse model provided new insights into the pathogenesis of neurodegenerative diseases, such as PCH10, caused by genetic mutations in tRNA metabolism-related molecules.

Polish wheat (Triticum polonicum) is a unique tetraploid wheat species characterized by an elongated outer glume. The genetic control of the long-glume trait by a single semi-dominant locus, P1 (from P olish wheat), was established more than 100 years ago, but the underlying causal gene and molecular nature remain elusive. Here, we report the isolation of VRT-A2, encoding an SVP-clade MADS-box transcription factor, as the P1 candidate gene. Genetic evidence suggests that in T. polonicum, a naturally occurring sequence rearrangement in the intron-1 region of VRT-A2 leads to ectopic expression of VRT-A2 in floral organs where the long-glume phenotype appears. Interestingly, we found that the intron-1 region is a key ON/OFF molecular switch for VRT-A2 expression, not only because it recruits transcriptional repressors, but also because it confers intron-mediated transcriptional enhancement. Genotypic analyses using wheat accessions indicated that the P1 locus is likely derived from a single natural mutation in tetraploid wheat, which was subsequently inherited by hexaploid T. petropavlovskyi. Taken together, our findings highlight the promoter-proximal intron variation as a molecular basis for phenotypic differentiation, and thus species formation in Triticum plants.

Neoantigen peptides arising from genetic alterations may serve as targets for personalized cancer vaccines and as positive predictors of response to immune checkpoint therapy. Mutations in genes regulating RNA splicing are common in hematological malignancies leading to dysregulated splicing and intron retention (IR). In this study, we investigated IR as a potential source of tumor neoantigens in multiple myeloma (MM) patients and the relationship of IR-induced neoantigens (IR-neoAg) with clinical outcomes. MM-specific IR events were identified in RNA-sequencing data from the Multiple Myeloma Research Foundation CoMMpass study after removing IR events that also occurred in normal plasma cells. We quantified the IR-neoAg load by assessing IR-induced novel peptides that were predicted to bind to major histocompatibility complex (MHC) molecules. We found that high IR-neoAg load was associated with poor overall survival in both newly diagnosed and relapsed MM patients. Further analyses revealed that poor outcome in MM patients with high IR-neoAg load was associated with high expression levels of T-cell co-inhibitory molecules and elevated interferon signaling activity. We also found that MM cells exhibiting high IR levels had lower MHC-II protein abundance and treatment of MM cells with a spliceosome inhibitor resulted in increased MHC-I protein abundance. Our findings suggest that IR-neoAg may represent a novel biomarker of MM patient clinical outcome and further that targeting RNA splicing may serve as a potential therapeutic strategy to prevent MM immune escape and promote response to checkpoint blockade.

Circular RNAs (circRNAs) represent a large group of non-coding RNAs that are widely detected in mammalian cells. Although most circRNAs are generated in a sense orientation, there is a group of circRNAs that are synthesized in an antisense orientation. High-throughput analysis of breast cancer specimens revealed a significant enrichment of 209 antisense circRNAs. The tumor suppressor SCRIB was shown to potentially produce thirteen circRNAs, three of which are in an antisense orientation. Among these three circRNAs, circSCRIB (hsa_circ_0001831) was the most enriched in the breast cancer panel. This antisense SCRIB circRNA was shown to span one intron and two exons. We hypothesized that this circRNA could decrease pre-mRNA splicing and mRNA translation. To test this, we generated a hsa_circ_0001831 expression construct. We found that there was decreased SCRIB mRNA production but increased cancer cell proliferation, migration, and invasion. In comparison, an exonic sequence construct did not affect mRNA splicing but decreased protein translation, leading to increased E-cadherin expression and decreased expression of N-cadherin and vimentin. Thus, there was increased cell migration, invasion, proliferation, colony formation, and tumorigenesis. Our study suggests a novel modulatory role of antisense circRNAs on their parental transcripts. This may represent a promising approach for developing circRNA-directed therapy.

Nature has long been a source of chemical diversity and its untapped potential is a major resource for solving our medical and energy problems. In particular, filamentous fungi have been prolific producers of medicinal compounds in the fight against human disease. Therefore, there has been interest in leveraging the advances in genomics to discover new fungal biosynthetic pathways that yield novel bioactive compounds. Heterologous expression of biosynthetic genes in model organisms will be increasingly necessary for high-throughput exploration of this genomic sequence space. Unfortunately, fungal genes contain many non-coding introns, which are difficult to manually annotate or predict in silico. Additionally, it is not possible to obtain intron-free cDNA from uncultivable species or transcriptionally silent gene clusters. This intron problem magnifies as the number of genomes increases and it risks derailing heterologous expression of this new genetic data. Unfortunately, the native spliceosome of the commonly used model eukaryote Saccharomyces cerevisiae cannot remove introns from distant fungi. In this thesis, I will describe my efforts to engineer S. cerevisiae with expanded spliceosome functionality. I identified two failure modes that prevent splicing of an intron from Aspergillus fumigatus. This led to the generation of a chimeric yeast-fungal BranchBinding Protein that has enhanced specificity for an intron containing a fungal branchpoint site. Expression of this mutant protein enabled a 2-fold improvement in splicing of an intron with a suboptimal branchpoint site. Additionally, we identified multiple synergistic splicing factor mutations with mBBP, YHC1-D36A and downregulation of IFH1, that enabled a 1.6-fold improvement of splicing of the A. fumigatus intron. Additional studies modifying the U2 small nuclear RNA as well splicing proofreaders PRP5, PRP16, and PRP28 will be described, highlighting the drawbacks of these approaches. This study is the first demonstration of improved splicing of an Aspergillus intron through spliceosome engineering in S. cerevisiae. Using the tools, methodologies, and yeast strains provided by this work, the spliceosome can be engineered with new function, broadening the scope of how synthetic biology will be used to enhance heterologous expression in diverse research fields, such as in the elucidation of the splicing code and in natural products discovery.

The physiological significance of a large family of heat-shock proteins (HSPs), comprised of the cytosolic HSP90A and the endoplasmic reticulum component of HSPB, is evident in prokaryotes and eukaryotes. The HSP90A is believed to play critical roles in diverse physiological functions of cell viability and chromosomal stability including stress management. Heightened abundance of hsp90β transcript was documented in Channa striatus, a freshwater fish, which is capable of surviving within an extremely hypoxic environment. To better understand the mechanism of hsp90β gene expression, we investigated its genomic organization. Eleven exons were identified, including a long upstream intron with a remarkable similarity with human, but not with chicken counterpart. Dual-luciferase assays identified promoter activity in a 1366 bp 5′-flanking segment beyond the transcription initiation site. Examination detected a minimal promoter of 754 bp containing a TATA-box, CAAT-enhancer in addition to providing clues regarding other enhancer and repressor elements. The driving capability of this minimal promoter was further validated by its binding ability with TATA-box binding protein and the generation of GFP expressing transgenic zebrafish (F2). Further, deletion of an inverted HIF (hypoxia inducible factor) motif RCGTG (upstream of the TATA-box) dramatically reduced luciferase expression in a hypoxic environment (CoCl2 treated cultivable cells) and was identified as a cis-acting HIF responsive element, necessary for the hypoxia-induced expression. The results obtained herein provide an insight regarding how hsp90β gene expression is controlled by HIF responsive element in teleost both during hypoxia stress management and normal physiological functions, and suggested that the hsp90β gene promoter could be used as a potential candidate for generating ornamental and food-fish transgenics.

The mitochondrial genome of Ophiostoma himal-ulmi, a species endemic to the Western Himalayas and one of the fungi that cause Dutch elm disease, has been sequenced and characterized. The mitochondrial genome was compared with other available genomes for members of the Ophiostomatales, including other agents of Dutch elm disease (Ophiostoma ulmi, Ophiostoma novo-ulmi subspecies novo-ulmi, and Ophiostoma novo-ulmi subspecies americana), and it was observed that gene synteny is highly conserved, and variability among members of the fungi that cause Dutch-elm disease is primarily due to the number of intron insertions. Among the fungi that cause Dutch elm disease that we examined, O. himal-ulmi has the largest mitochondrial genomes (ranging from 94 934 to 111 712 bp), owing to the expansion of the number of introns.

Cystic fibrosis (CF) is a rare autosomal recessive disease with only one pathogenic gene cystic fibrosis transmembrane conductance regulator (CFTR). To identify the potential pathogenic mutations in a Chinese patient with CF, we conducted Sanger sequencing on the genomic DNA of the patient and his parents and detected all 27 coding exons of CFTR and their flanking intronic regions. The patient is a compound heterozygote of c.2909G > A, p.Gly970Asp in exon 18 and c.1210-3C > G in cis with a poly-T of 5T (T5) sequence, 3 bp upstream in intron 9. The splicing effect of c.1210-3C > G was verified via minigene assay in vitro, indicating that wild-type plasmid containing c.1210-3C together with T7 sequence produced a normal transcript and partial exon 10-skipping-transcript, whereas mutant plasmid containing c.1210-3G in cis with T5 sequence caused almost all mRNA to skip exon 10. Overall, c.1210-3C > G, the newly identified pathogenic mutation in our patient, in combination with T5 sequence in cis, affects the CFTR gene splicing and produces nearly no normal transcript in vitro. Moreover, this patient carries a p.Gly970Asp mutation, thus confirming the high-frequency of this mutation in Chinese patients with CF.

Different toxins acting on Kv1.3 channel have been isolated from animal venom. MeuKTX toxin from Mesobuthus eupeus phillipsi scorpion and shtx-k toxin from Stichodactyla haddoni sea anemone have been identified as two effective Kv1.3 channel blockers. In this work, we characterized the genomic organization of both toxins. MeuKTX gene contains one intron and two exons, similar to the most scorpion toxins. There are a few reports of genomic structure of sea anemone toxins acting on Kv channels. The sequence encoding mature peptide of shtx-k was located in an exon separated by an intron from the coding exon of the propeptide and signal region. In order to make a peptide with more affinity for Kv1.3 channel and greater stability, the shtx-k/ MeuKTX chimeric peptide was designed and constructed using splicing by overlap extension-PCR (SOE-PCR) method. MeuKTX, shtx-k, and shtx-k/MeuKTX were cloned and the expression of the soluble proteins in E. coli was determined. Molecular docking studies indicated more inhibitory effect of shtx-k/MeuKTX on Kv1.3 channel compared to shtx-k and MeuKTX toxins. Key amino acids binding channel from both toxins, also involved in interaction of chimeric peptide with channel. Our results showed that the fusion peptide, shtx-k/MeuKTX could be an effective agent to target Kv1.3 channel.

Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this workflow circumvents issues related to inadequate reference annotations and allows for incorporation of short-read sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long noncoding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing data sets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene.

CircRNAs are a large class of endogenous single-stranded RNA that is different from other linear RNA, which are produced by back-splicing and fusion of either exons, introns, or both exon-intron into covalently closed loops. CircRNAs are found in almost all living organisms and have emerged as potentially important players effecting on all life activities. It was characterized by stable structure, resistant to RNA degradation, highly abundance and conservation and tissue-specific expression. Early circRNAs were ignored as a by-product of meaningless abnormally cut RNA and had little biological function. Currently, circRNAs have become a research hotspot due to its special characteristics. CircRNAs could function as miRNA sponges, interfere with splicing and bind to protein to regulate the expression of parental genes and so on. In recent years, an increasing number of studies have revealed that circRNAs are closely related to a series of physiological and pathological processes. Additionally, circRNAs play an important role in the occurrence and development of a variety of diseases, suggesting circRNAs may be as novel indicators or biomarkers for cancer and other diseases with which they are associated. In this article, we review the biogenesis, biological functions of circRNAs and recent advances in circRNAs research in human diseases. Results will provide new insights on the roles and new ideas of circRNAs for the diagnosis and treatment of diseases and possible directions and approach for future circRNA applications.

During spliceosome assembly, interactions that bring the 5′ and 3′ ends of an intron in proximity are critical for the production of mature mRNA. Here, we report synergistic roles for the stem-loops 3 (SL3) and 4 (SL4) of the human U1 small nuclear RNA (snRNA) in maintaining the optimal U1 snRNP function, and formation of cross-intron contact with the U2 snRNP. We find that SL3 and SL4 bind distinct spliceosomal proteins and combining a U1 snRNA activity assay with siRNA-mediated knockdown, we demonstrate that SL3 and SL4 act through the RNA helicase UAP56 and the U2 protein SF3A1, respectively. In vitro analysis using UV crosslinking and splicing assays indicated that SL3 likely promotes the SL4-SF3A1 interaction leading to enhancement of A complex formation and pre-mRNA splicing. Overall, these results highlight the vital role of the distinct contacts of SL3 and SL4 in bridging the pre-mRNA bound U1 and U2 snRNPs during the early steps of human spliceosome assembly.

The second most common form of Charcot-Marie-Tooth neuropathy (CMT), X-linked CMT type X1 (CMTX1), is caused by coding and non-coding mutations in the gap junction beta 1 (GJB1) gene. The non-coding GJB1 c.-103C > T mutation (NM_000166.5) has been reported to cause CMTX1 in multiple families. This study assessed the internal ribosomal entry site (IRES) activity previously reported for the rat Gjb1 P2 5’ untranslated region (UTR). Using a bicistronic assay and transfecting RT4 Schwann cells, IRES activity of the human GJB1 P2 5’ UTR was compared to the GJB1 P2 5’ UTR containing either the c.-103C > T mutation or the non-pathogenic c.-102G > A variant. No differences in GJB1 P2 5’ UTR IRES activity were observed between the negative control, the wild-type P2 5’ UTR, the c.-103C > T 5’ UTR or the c.-102G > A 5’ UTR, irrespective of the GJB1 intron being present (p = .429 with intron, and p = .865 without). A theoretical c.-131A > G variant was predicted to result in the same RNA secondary structure as the GJB1 c.-103C > T P2 5’ UTR. However, no significant difference was observed between expression from the wild-type GJB1 P2 5’ UTR and the GJB1 c.-131A > G variant (p = .688). Deletion of the conserved region surrounding the c.-103C > T mutation (c.-108_-103del) resulted in significantly higher expression than the c.-103C > T mutation alone (p = .019), suggesting that the conserved c.-108_-103 region was not essential for translation. The reporter assays in this study do not recapitulate the previously reported GJB1 IRES activity and suggest an alternate pathogenic mechanism for the c.-103C > T CMTX1 non-coding mutation.

Lithium (Li), valproate (VPA) and lamotrigine (LTG) are commonly used to treat bipolar disorder (BD). While their clinical efficacy is well established, the mechanisms of action at the molecular level are still incompletely understood. Here we investigated the molecular effects of Li, LTG and VPA treatment in induced pluripotent stem cell (iPSC)-derived neural precursor cells (NPCs) generated from 3 healthy controls (CTRL), 3 affective disorder Li responsive patients (Li-R) and 3 Li non-treated patients (Li-N) after 6 h and 1 week of exposure. Differential expression (DE) analysis after 6 h of treatment revealed a transcriptional signature that was associated with all three drugs and most significantly enriched for ribosome and oxidative phosphorylation (OXPHOS) pathways. In addition to the shared DE genes, we found that Li exposure was associated with 554 genes uniquely regulated in Li-R NPCs and enriched for spliceosome, OXPHOS and thermogenesis pathways. In-depth analysis of the treatment-associated transcripts uncovered a significant decrease in intron retention rate, suggesting that the beneficial influence of these drugs might partly be related to splicing. We examined the mitochondrial respiratory function of the NPCs by exploring the drugs’ effects on oxygen consumption rate (OCR) and glycolytic rate (ECAR). Li improved OCR levels only in Li-R NPCs by enhancing maximal respiration and reserve capacity, while VPA enhanced maximal respiration and reserve capacity in Li-N NPCs. Overall, our findings further support the involvement of mitochondrial functions in the molecular mechanisms of mood stabilizers and suggest novel mechanisms related to the spliceosome, which warrant further investigation.

Walnut is a popular nut tree species and usually suffers from drought stress. However, little information is available on the mechanism of walnut responding to drought stress, resulting in lack of basic understanding for its resistance. In order to excavate more functional genes that can respond to stressors, and enrich the theoretical basis for walnut resistance, in this study, 5 MYB genes with complete ORFs were identified from J. regia and the basic bio-information as well as expression patterns in different tissues and response to drought and ABA stresses were confirmed using qRT-PCR assay. The results showed that 2 JrMYB genes belong to R1-MYB subfamily and 3 JrMYBs belong to R2R3-MYB, encoding the proteins from 212 to 362 aa in length. The phylogenetic analysis categorized proteins of 5 JrMYBs and 40 Arabidopsis AtMYBs into 10 subgroups. JrMYBs in the same subgroup exhibited significant similarities in the composition of conserved domains and motifs in amino acid sequences and exon/intron organization in DNA sequences. The results of qRT-PCR analysis revealed that JrMYB genes diversely expressed in various tissues. Moreover, the expression values of JrMYBs were upregulated or downregulated significantly under drought and ABA stresses. Most attractively, in contrast with suffering from drought stress alone, the treatments with drought and additional ABA greatly enhanced the transcript levels of JrMYBs. All these results suggested that JrMYB genes play a vital role in plant biological processes and drought as well as ABA stress response, and possibly perform as ABA-dependent drought response transcription factors in plant.

MYB transcription factors play pivotal roles in hormone conduction signaling and abiotic stress response. In this study, 54 differentially expressed ZmMYB genes were identified and comprehensive analyses were conducted including gene’s structure, chromosomal localization, phylogenetic tree, motif prediction, cis-elements and expression patterns. The results showed that 54 genes were unevenly distributed on 10 chromosomes and classified into eleven main subgroups by phylogenetic analysis, supported by motif and exon/intron analyses. The mainly stress-related cis-elements were ABRE, ARE, MBS and DRE-core. In addition, 8 core ZmMYB genes were identified by co-expression network. qRT-PCR results showed that the 8 ZmMYB genes exhibited different expression levels under different abiotic stresses, indicating that they were responsive to various abiotic stress. These results will provide insight for further functional investigation of ZmMYB genes.

RNA alternative splicing (AS) is prevalent in higher organisms and plays a paramount role in biology; therefore, it is crucial to have comprehensive knowledge on AS to understand biology. However, knowledge is limited about how AS activates in a single plant and functions in a biological process. Ginseng is one of the most widely used medicinal herbs that is abundant in a number of medicinal bioactive components, especially ginsenosides. In this study, we sequenced the transcripts of 14 organs from a 4-year-old ginseng plant and quantified their ginsenoside contents. We identified AS genes by analyzing their transcripts with the ginseng genome and verified their AS events by PCR. The plant had a total of 13,863 AS genes subjected to 30,801 AS events with five mechanisms: skipped exon, retained intron, alternative 5'splice site, alternative 3' splice site, and mutually exclusive exon. The genes that were more conserved, had more exons, and/or expressed across organs were more likely to be subjected to AS. AS genes were enriched in over 500 GO terms in the plant even though the number of AS gene-enriched GO terms varied across organs. At least 24 AS genes were found to be involved in ginsenoside biosynthesis. These AS genes were significantly up-enriched and more likely to form a co-expression network, thus suggesting the functions of AS and correlations of the AS genes in the process. This study provides comprehensive insights into the molecular characteristics and biological functions of AS in a single plant; thus, helping better understand biology.

Pre-mRNA splicing is carried out by the spliceosome and involves splice site recognition, removal of introns, and ligation of exons. Components of the spliceosome have been shown to interact with the elongating RNA polymerase II (RNAPII), which is thought to allow splicing to occur concurrently with transcription. However, little is known about the regulation and efficiency of cotranscriptional splicing in human cells. In this study, we used Bru-seq and BruChase-seq to determine the cotranscriptional splicing efficiencies of 17,000 introns expressed across six human cell lines. We found that less than half of all introns across these six cell lines were cotranscriptionally spliced. Splicing efficiencies for individual introns showed variations across cell lines, suggesting that splicing may be regulated in a cell type–specific manner. Moreover, the splicing efficiency of introns varied within genes. The efficiency of cotranscriptional splicing did not correlate with gene length, intron position, splice site strengths, or the intron/neighboring exons GC content. However, we identified binding signals from multiple RNA-binding proteins (RBPs) that correlated with splicing efficiency, including core spliceosomal machinery components—such as SF3B4, U2AF1, and U2AF2 showing higher binding signals in poorly spliced introns. In addition, multiple RBPs, such as BUD13, PUM1, and SND1, showed preferential binding in exons that flank introns with high splicing efficiencies. The nascent RNA splicing patterns presented here across multiple cell types add to our understanding of the complexity in RNA splicing, wherein RNA-binding proteins may play important roles in determining splicing outcomes in a cell type– and intron-specific manner.

T cells can be subdivided into a number of different subsets that are defined by their distinct functions. While the specialization of different T cell subsets is partly achieved by the expression of specific genes, the overall transcriptional profiles of all T cells appear very similar. Alternative mRNA splicing is a mechanism that facilitates greater transcript/protein diversity from a limited number of genes, which may contribute to the functional specialization of distinct T cell subsets. In this study we employ a combination of short-read and long-read sequencing technologies to compare alternative mRNA splicing between the CD4 and CD8 T cell lineages. While long-read technology was effective at assembling full-length alternatively spliced transcripts, the low sequencing depth did not facilitate accurate quantitation. On the other hand, short-read technology was ineffective at assembling full-length transcripts but was highly accurate for quantifying expression. We show that integrating long-read and short-read data together achieves a more complete view of transcriptomic diversity. We found that while the overall usage of transcript isoforms was very similar between the CD4 and CD8 lineages, there were numerous alternative spliced mRNA isoforms that were preferentially used by one lineage over the other. These alternative spliced isoforms included ones with different exon usage, exon exclusion or intron inclusion, all of which are expected to significantly alter the protein sequence.

Toll-like receptors (TLRs) play a crucial role in the innate immune system, which is the first line of defence against pathogens and pathogenic products in fish. In the present study, we cloned the full-length cDNA and genome sequences of two TLR13 s (OnTLR13a, OnTLR13b) from Nile tilapia (Oreochromis niloticus). TLR family motifs, i.e., the leucine-rich repeat (LRR) domains and Toll/interleukin (IL)-1 receptor (TIR) domains, were conserved in the putative proteins OnTLR13a and OnTLR13b, with fifteen LRR domains and one TIR domain. Four exons and three introns were identified in the OnTLR13a genome sequence, and three exons and two introns were identified in the OnTLR13b genome sequence. In healthy Nile tilapia tissues, OnTLR13a and OnTLR13b were ubiquitously expressed in all 11 tested tissues/organs. The highest expression levels were observed in the spleen (OnTLR13a) and blood (OnTLR13b), and the lowest expression levels were observed in the liver (OnTLR13a) and stomach (OnTLR13b). The expression level of OnTLR13b at 5.5 days postfertilization (dpf) was significantly higher than that at the other 8 time points (2.5, 3.5, 4.5, 5, 6, 6.5, 7.5 and 8.5 dpf). Upon stimulation with an intraperitoneal injection of 200 μL (107 CFU/mL) Streptococcus agalactiae, the expression levels of OnTLR13a and OnTLR13b were significantly upregulated in the intestine and gill. After cotransfection with MyD88, OnTLR13a significantly increased MyD88-dependent NF-κB activation in 293 T cells. However, OnTLR13b significantly impaired MyD88-dependent NF-κB activation. In addition, TLR13a slightly increased MyD88-dependent AP-1 activation, and TLR13b significantly increased MyD88-dependent AP-1 activation. TLR13a significantly increased MyD88-dependent interferon-β (IFN-β) activation, and TLR13b had no effect on MyD88-dependent IFN-β activation. These findings suggest that although the deduced protein structure of OnTLR13 is evolutionarily conserved between OnTLR13 and other TLR members, its signal transduction function is markedly different. Co-immunoprecipitation (Co-IP) assays showed that both OnTLR13a and OnTLR13b could interact with OnMyD88. RNA pulldown assays showed that TLR13a and TLR13b could combine with the 23S rRNA of S. agalactiae. These results indicate that TLR13a and TLR13b play important roles in the innate immune response against bacterial infection in Nile tilapia.

Recent studies suggest that Poly(ADP-ribose) polymerase 1 (PARP1) acts as an RNA-binding protein in a majority of renal diseases with tubular cell injury. However, detailed knowledge of RNA targets and the RNA-binding regions for PARP1 is unknown. Herein, mapping of iRIP-seq reads in HK-2 renal tubular epithelial cells showed a biased distribution at coding sequence (CDS) and intron regions that is specific to these cells. A total of 1708 differentially expressed genes were identified after PARP1 knockdown using RNA-seq. Furthermore, transcriptome analysis also showed that selective variable splicing was globally regulated by PARP1 in HK-2 cells. By comparison of PARP1 RNA-seq and iRIP-seq data, we found 68 overlapping genes that are enriched in 'extracellular matrix' pathway. Follow-up identification of their interactions may contribute vital insights into the regulatory role of PARP1 as an RNA-binding protein in HK-2 cells.

Background Group II introns are mobile genetic elements used as efficient gene targeting tools. They function as both ribozymes and retroelements. Group IIC introns are the only class reported so far to be associated with integrons. In order to identify group II introns linked with integrons and CALINS (cluster of attC sites lacking a neighboring integron integrase) within halophiles, we mined for integrons in 28 assembled metagenomes from hypersaline environments and publically available 104 halophilic genomes using Integron Finder followed by blast search for group II intron reverse transcriptases (RT)s. Results We report the presence of different group II introns associated with integrons and integron-related sequences denoted by UHB.F1, UHB.I2, H.ha.F1 and H.ha.F2. The first two were identified within putative integrons in the metagenome of Tanatar-5 hypersaline soda lake, belonging to IIC and IIB intron classes, respectively at which the first was a truncated intron. Other truncated introns H.ha.F1 and H.ha.F2 were also detected in a CALIN within the extreme halophile Halorhodospira halochloris, both belonging to group IIB introns. The intron-encoded proteins (IEP) s identified within group IIB introns belonged to different classes: CL1 class in UHB.I2 and bacterial class E in H.ha.Fa1 and H.ha.F2. A newly identified insertion sequence (ISHahl1) of IS200/605 superfamily was also identified adjacent to H. halochloris CALIN. Finally, an abundance of toxin-antitoxin (TA) systems was observed within the identified integrons. Conclusion So far, this is the first investigation of group II introns within integrons in halophilic genomes and metagenomes from hypersaline environments. We report the presence of group IIB introns associated with integrons or CALINs. This study provides the basis for understanding the role of group IIB introns in the evolution of halophiles and their potential biotechnological role.

Self-cleaving ribozymes are genetic elements found in all domains of life, but their evolution remains poorly understood. A ribozyme located in the second intron of the cytoplasmic polyadenylation binding protein 3 gene (CPEB3) shows high sequence conservation in mammals, but little is known about the functional conservation of self-cleaving ribozyme activity across the mammalian tree of life or during the course of mammalian evolution. Here, we use a phylogenetic approach to design a mutational library and a deep sequencing assay to evaluate the in vitro self-cleavage activity of numerous extant and resurrected CPEB3 ribozymes that span over 100 My of mammalian evolution. We found that the predicted sequence at the divergence of placentals and marsupials is highly active, and this activity has been conserved in most lineages. A reduction in ribozyme activity appears to have occurred multiple different times throughout the mammalian tree of life. The in vitro activity data allow an evaluation of the predicted mutational pathways leading to extant ribozyme as well as the mutational landscape surrounding these ribozymes. The results demonstrate that in addition to sequence conservation, the self-cleavage activity of the CPEB3 ribozyme has persisted over millions of years of mammalian evolution.

Previous evolutionary reconstructions have concluded that early eukaryotic ancestors including both the last common ancestor of eukaryotes and of all fungi had intron-rich genomes. By contrast, some extant eukaryotes have few introns, underscoring the complex histories of intron–exon structures, and raising the question as to why these few introns are retained. Here, we have used recently available fungal genomes to address a variety of questions related to intron evolution. Evolutionary reconstruction of intron presence and absence using 263 diverse fungal species supports the idea that massive intron reduction through intron loss has occurred in multiple clades. The intron densities estimated in various fungal ancestors differ from zero to 7.6 introns per 1 kb of protein-coding sequence. Massive intron loss has occurred not only in microsporidian parasites and saccharomycetous yeasts, but also in diverse smuts and allies. To investigate the roles of the remaining introns in highly-reduced species, we have searched for their special characteristics in eight intron-poor fungi. Notably, the introns of ribosome-associated genes RPL7 and NOG2 have conserved positions; both intron-containing genes encoding snoRNAs. Furthermore, both the proteins and snoRNAs are involved in ribosome biogenesis, suggesting that the expression of the protein-coding genes and noncoding snoRNAs may be functionally coordinated. Indeed, these introns are also conserved in three-quarters of fungi species. Our study shows that fungal introns have a complex evolutionary history and underappreciated roles in gene expression.

Yishui Lily 140 (Lilium lancifolium) is a hybrid lily species which was bred from wild lily varieties due to its edible and medicinal value. In this study, we have sequenced the complete chloroplast (cp) of L. lancifolium. The complete cp sequence is 152,643 bp long, with a large single copy (LSC) region of 82,084 bp, a small single copy (SSC) region of 17,513 bp, and two inverted repeat (IR) regions of 26,492 bp each. The GC contents of the complete cp genomes are 37.0%. It contains 132 genes, including 86 coding genes, 8 ribosomal RNAs, and 38 transfer RNAs. Among them, 16 different genes have a single intron and the remaining two genes have double introns, including nine cis-splicing and one trans-splicing genes. Compared with other species, we found three high variation hot spots and 96 repeats sequence. The genetic information of Lilium can be enriched as well as identifying proximal species. They are edible and have medicinal value for humans. Therefore, sequencing of Yishui Lily 140 is important to explore the cp genome composition.

Lilaeopsis chinensis (L.) Kuntze is a traditional Chinese medicinal plant for treating diuresis, abdominal pain and eczema. In this study, its complete chloroplast genome was assembled from the whole genome Illumina sequencing data. The circular genome is 153,162 bp long, and comprises a pair of inverted repeat regions (IRs, 25,072 bp each), a large single-copy region (LSC, 84,288 bp) and a small single-copy region (SSC, 18,730 bp). It encodes a total of 113 genes (79 protein-coding, 30 tRNA and 4 rRNA genes), with 19 of them occurring in double copies. Introns were detected in 11 protein coding genes (PCG) and 6 tRNA genes. The nucleotide composition is asymmetric (30.9% A, 19.2% C, 18.4% G and 31.5% T) with an overall A + T content of 62.4%. Phylogenetic analysis challenged the traditional taxonomic framework of the family Apiaceae, and indicated that Lilaeopsis chinensis (L.) Kuntze is closely related to Hydrocotyle verticillata.

Ulva intestinalis Linnaeus 1753 (Ulvophyceae, Chlorophyta) is a marine green macroalga that is distributed on coasts of the Yellow Sea and the Bohai Sea in China. Here, the complete chloroplast genome of U. intestinalis was constructed and analyzed comparatively. The chloroplast genome of U. intestinalis is a 99,041-bp circular molecule that harbors a total of 112 genes including 71 protein-coding genes (PCGs), 26 transfer RNA genes (tRNAs), three ribosomal RNA genes (rRNAs), three free-standing open reading frames (orfs) and nine intronic orfs, and ten introns in seven genes (atpA, infA, psbB, psbC, petB, rrnL, and rrnS). The maximum likelihood (ML) phylogenomic analysis shows that U. intestinalis firstly groups with Ulva compressa, and then these two species together with the Ulva australis–Ulva fenestrata–Ulva rotundata subclade form a monophyletic clade, Ulva lineage II. U. intestinalis chloroplast genome is the only one in Ulva lineage II where the reversal of a collinear block of two genes (psbD–psbC) did not occur, and its genome structure is consistent with that of most chloroplast genomes in Ulva lineage I, indicating that the similarity of genome structure is not completely related to the genetic relationship of Ulva species. Our genomic data will facilitate the development of specific high-resolution chloroplast molecular markers for rapid identification of U. intestinalis, and help us understand its population diversity and genetic characteristics on a global scale.

Introduction The BCR/ABL1 fusion exists in several different isoforms, depending on the precise position of the t(9;22)(q34;q11) genomic breakpoints in each case. In chronic myeloid leukemia (CML), the two most common isoforms are known as e13a2 and e14a2, which are shorthand for mRNA fusions of BCR exon 13 or BCR exon 14, respectively, to ABL1 exon 2. Historically, these two fusions were referred to as b2a2 and b3a2, with b2 (BCR exon 13) and b3 (BCR exon 14) corresponding to the second and third exons within the classically-defined 5.8-kb major breakpoint cluster region (M-BCR) within BCR.1 Both e13a2 and e14a2 encode a 210-kDa BCR/ABL1 protein (p210), but e14a2 encodes a chimeric protein that is 25 amino acids larger than e13a2, corresponding to the 75 bp of BCR exon 14. At the genomic DNA level, the chromosome 22 breakpoint typically occurs in BCR intron 13 for e13a2 and BCR intron 14 for e14a2. Collectively, e13a2 and e14a2 BCR/ABL1 account for 98% of CML cases, with the majority of these expressing e14a2. Approximately 10% of cases express both isoforms.1,2 A large international survey of CML cases found small but significant differences in the relative prevalence of the two fusions in relation to age and gender, with e13a2 being more frequent in males and less common in the elderly.2

Interleukin 7 receptor α-chain is crucial for the development and maintenance of T cells and is genetically associated with autoimmune disorders including multiple sclerosis (MS), a demyelinating disease of the CNS. Exon 6 of IL7R encodes for the transmembrane domain of the receptor and is regulated by alternative splicing: inclusion or skipping of IL7R exon 6 results in membrane-bound or soluble IL7R isoforms, respectively. We previously identified a SNP (rs6897932) in IL7R exon 6, strongly associated with MS risk and showed that the risk allele (C) increases skipping of the exon, resulting in elevated levels of sIL7R. This has important pathological consequences as elevated levels of sIL7R has been shown to exacerbate the disease in the experimental autoimmune encephalomyelitis mouse model of MS. Understanding the regulation of exon 6 splicing provides important mechanistic insights into the pathogenesis of MS. Here we report two mechanisms by which IL7R exon 6 is controlled. First, a competition between PTBP1 and U2AF2 at the polypyrimidine tract (PPT) of intron 5, and second, an unexpected U2AF2-mediated assembly of spicing factors in the exon. We noted the presence of a branchpoint sequence (BPS) (TACTAAT or TACTAAC) within exon 6, which is stronger with the C allele. We also noted that the BPS is followed by a PPT and conjectured that silencing could be mediated by the binding of U2AF2 to that tract. In support of this model, we show that evolutionary conservation of the exonic PPT correlates well with the degree of alternative splicing of exon 6 in two non-human primate species and that U2AF2 binding to this PPT recruits U2 snRNP components to the exon. These observations provide the first explanation for the stronger silencing of IL7R exon 6 with the disease associated C allele at rs6897932.

Zinc finger-homeodomain (ZF-HD) proteins encode a family of plant-specific transcription factors that play essential roles in regulating plant growth and development as well as responses to abiotic/biotic stresses by activating or repressing the target genes. In this study, genome-wide characterization and expression profiling of the ZF-HD gene family in cucumber (Cucumis sativus) were performed for the first time. By using bioinformatics approaches, a total of 13 ZF-HD genes (designated as CsMIF1-CsMIF3 and CsZHD1-CsZHD10) were identified in the cucumber genome, which were unevenly distributed on six chromosomes. According to the phylogenetic analysis of cucumber and other species, they were divided into two distinct families, MINI ZINC FINGER (MIF) and zinc finger-homeodomain (ZHD), and the ZHD family was further divided into six subfamilies (ZHDI-ZHDVI). CsZF-HD members were mostly conserved in each subfamily with minor variations in motif distribution, and gene structure analysis showed that the CsZF-HD genes had only one intron or no intron at all. Expression analysis showed that most CsZF-HD genes had tissue-specific expression patterns, and some of them exhibited highly variable expression during fruit development. qRT-PCR results indicated that the selected CsZF-HD genes were responsive to drought stress, and some of them were differentially expressed in response to the inoculation of powdery mildew (PM) and downy mildew (DM) based on publicly available RNA-seq data. The results lay the foundation for further functional analysis of the ZF-HD genes and explore their potential application to the improvement of stress tolerance in cucumber.