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De novo mutations affecting the pre-mRNA splicing factor U2AF2 are associated with developmental delays and intellectual disabilities, yet the molecular basis is unknown. Here, we demonstrated by fluorescence anisotropy RNA binding assays that recurrent missense mutants (Arg149Trp, Arg150His, or Arg150Cys) decreased the binding affinity of U2AF2 for a consensus splice site RNA. Crystal structures at 1.4 Å resolutions showed that Arg149Trp or Arg150His disrupted hydrogen bonds between U2AF2 and the terminal nucleotides of the RNA site. Reanalysis of publicly available RNaseq data confirmed that U2AF2 depletion altered splicing of transcripts encoding RNA binding proteins (RBPs). These results confirmed that the impaired RNA interactions of Arg149Trp and Arg150His U2AF2 variants could contribute to dysregulating an RBP-governed neurodevelopmental program of alternative splicing.

Dysregulation of the flow of information from genomic DNA to RNA to protein occurs within all cancer types. In this review, we described the current state of understanding of how RNA processing is dysregulated in cancer with a focus on mutations in the RNA splicing factor machinery that are highly prevalent in hematologic malignancies. We discuss the downstream effects of these mutations highlighting both individual genes as well as common pathways that they perturb. We highlight examples of how alterations in RNA processing have been harnessed for therapeutic intent as well as to promote the selective toxicity of cancer cells., (© 2024 Biswas et al.)

The two transesterification reactions of pre-mRNA splicing require highly complex yet well-controlled rearrangements of small nuclear RNAs and proteins (snRNP) in the spliceosome. The efficiency and accuracy of these reactions are critical for gene expression, as almost all human genes pass through pre-mRNA splicing. Key parameters that determine the splicing outcome are the length of the intron, the strengths of its splicing signals and gaps between them, and the presence of splicing controlling elements. In particular, the gap between the branchpoint (BP) and the 3' splice site (ss) of introns is a major determinant of the splicing efficiency. This distance falls within a small range across the introns of an organism. The constraints exist possibly because BP and 3'ss are recognized by BP-binding proteins, U2 snRNP and U2 accessory factors (U2AF) in a coordinated manner. Furthermore, varying distances between the two signals may also affect the second transesterification reaction since the intervening RNA needs to be accurately positioned within the complex RNP machinery. Splicing such pre-mRNAs requires cis-acting elements in the RNA and many trans-acting splicing regulators. Regulated pre-mRNA splicing with BP-distant 3'ss adds another layer of control to gene expression and promotes alternative splicing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Ribonuclease P (RNase P) plays a vital role in the maturation of tRNA across bacteria, archaea, and eukaryotes. However, how RNase P assembles various components to achieve specific cleavage of precursor tRNA (pre-tRNA) in different organisms remains elusive. In this study, we employed single-molecule fluorescence resonance energy transfer to probe the dynamics of RNase P from E. coli ( Escherichia coli ) and Mja ( Methanocaldococcus jannaschii ) during pre-tRNA cleavage by incorporating five Cy3-Cy5 pairs into pre-tRNA and RNase P. Our results revealed significant differences in the assembly and catalytic mechanisms of RNase P between E. coli and Mja at both the RNA and protein levels. Specifically, the RNA of E. coli RNase P ( Eco RPR) can adopt an active conformation that is capable of binding and cleaving pre-tRNA with high specificity independently. The addition of the protein component of E. coli RNase P (RnpA) enhances and accelerates pre-tRNA cleavage efficiency by increasing and stabilizing the active conformation. In contrast, Mja RPR is unable to form the catalytically active conformation on its own, and at least four proteins are required to induce the correct folding of Mja RPR. Mutation experiments suggest that the functional deficiency of Mja RPR arises from the absence of the second structural layer, and proper intermolecular assembly is essential for Mja RNase P to be functional over a broad temperature range. We propose models to illustrate the distinct catalytic patterns and RNA-protein interactions of RNase P in these two organisms., Competing Interests: Competing interests statement:The authors declare no competing interest.

The production of eukaryotic mRNAs requires transcription by RNA polymerase (pol) II and co-transcriptional processing, including capping, splicing, and cleavage and polyadenylation. Pol II can positively affect co-transcriptional processing through interaction of factors with its carboxyl terminal domain (CTD), comprising 52 repeats of the heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, and pol II elongation rate can regulate splicing. Splicing, in turn, can also affect transcriptional activity and transcription elongation defects are caused by some splicing inhibitors. Multiple small molecule inhibitors of splicing are now available, some of which specifically target SF3B1, a U2 snRNP component. SF3B1 inhibition results in a general downregulation of transcription elongation, including premature termination of transcription caused by increased use of intronic poly(A) sites. Here, we have investigated the effect of Madrasin and Isoginkgetin, two non-SF3B1 splicing inhibitors, on splicing and transcription. Surprisingly, we found that both Madrasin and Isoginkgetin affect transcription before any effect on splicing, indicating that their effect on pre-mRNA splicing is likely to be indirect. Both small molecules promote a general downregulation of transcription. Based on these and other published results, we conclude that these two small molecules should not be considered as primarily pre-mRNA splicing inhibitors., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tellier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Key Message: The Arabidopsis RNA helicase LOS4 plays a key role in regulating pre-mRNA splicing of the genes EIN2, CTR1, and ERS2 in ethylene signaling pathway. The plant hormone ethylene plays diverse roles in plant growth, development, and responses to stress. Ethylene is perceived by the membrane-bound ethylene receptors complex, and then triggers downstream components, such as EIN2, to initiate signal transduction into the nucleus, leading to the activation of ethylene-responsive genes. Over the past decades, substantial information has been accumulated regarding gene cloning, protein-protein interactions, and downstream gene expressions in the ethylene pathway. However, our understanding of mRNA post-transcriptional processing and modification of key genes in the ethylene signaling pathway remains limited. This study aims to provide evidence demonstrating the involvement of the Arabidopsis RNA helicase LOS4 in pre-mRNA splicing of the genes EIN2, CTR1, and ERS2 in ethylene signaling pathway. Various genetic approaches including RNAi gene silencing, CRISPR-Cas9 gene editing, and amino acid mutations were employed in this study. When LOS4 was silenced or knocked down, the ethylene sensitivity of etiolated seedlings was significantly enhanced. Further investigation revealed errors in the EIN2 pre-mRNA splicing when LOS4 was knocked down. In addition, aberrant pre-mRNA splicing was observed in the ERS2 and CTR1 genes in the pathway. Biochemical assays indicated that the los4-2 (E94K) mutant protein exhibited increased ATP binding and enhanced ATP hydrolytic activity. Conversely, the los4-1 (G364R) mutant had reduced substrate RNA binding and lower ATP binding activities. These findings significantly advanced our comprehension of the regulatory functions and molecular mechanisms of RNA helicase in ethylene signaling., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Proper gene expression requires the collaborative effort of multiple macromolecular machines to produce functional messenger RNA. As RNA polymerase II (RNA Pol II) transcribes DNA, the nascent pre-messenger RNA is heavily modified by other complexes such as 5' capping enzymes, the spliceosome, the cleavage, and polyadenylation machinery as well as RNA-modifying/editing enzymes. Recent evidence has demonstrated that pre-mRNA splicing and 3' end cleavage can occur on similar timescales as transcription and significantly cross-regulate. In this review, we discuss recent advances in co-transcriptional processing and how it contributes to gene regulation. We highlight how emerging areas-including coordinated splicing events, physical interactions between the RNA synthesis and modifying machinery, rapid and delayed splicing, and nuclear organization-impact mRNA isoforms. Coordination among RNA-processing choices yields radically different mRNA and protein products, foreshadowing the likely regulatory importance of co-transcriptional RNA folding and co-transcriptional modifications that have yet to be characterized in detail., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Poly (A) binding protein interacting protein 1 (PAIP1) has been shown to causally contribute to the development and progression of cancer. However, the mechanisms of the PAIP1 regulation in tumor cells remain poorly understood., Results: Here, we used a recently developed UV cross-linking and RNA immunoprecipitation method (iRIP-seq) to map the direct and indirect interaction sites between PAIP1 and RNA on a transcriptome-wide level in HeLa cells. We found that PAIP1 not only binds to 3'UTRs, but also to pre-mRNAs/mRNAs with a strong bias towards the coding region and intron. PAIP1 binding sites are enriched in splicing enhancer consensus GA-rich motifs. RNA-seq analysis revealed that PAIP1 selectively modulates the alternative splicing of genes in some cancer hallmarks including cell migration, the mTOR signaling pathway and the HIF-1 signaling pathway. PAIP1-regulated alternative splicing events were strongly associated with PAIP1 binding, demonstrating that the binding may promote selection of the nearby splice sites. Deletion of a PAIP1 binding site containing seven repeats of GA motif reduced the PAIP1-mediated suppression of the exon 6 inclusion in a VEGFA mRNA isoform. Proteomic analysis of the PAIP1-interacted proteins revealed the enrichment of the spliceosome components and splicing factors., Conclusions: These findings suggest that PAIP1 is both a polyadenylation and alternative splicing regulator, that may play a large role in RNA processing via its role in alternative splicing regulation., (© 2024. The Author(s).)

Nuclear localization of the metabolic enzyme PKM2 is widely observed in various cancer types. We identify nuclear PKM2 as a non-canonical RNA-binding protein (RBP) that specifically interacts with folded RNA G-quadruplex (rG4) structures in precursor mRNAs (pre-mRNAs). PKM2 occupancy at rG4s prevents the binding of repressive RBPs, such as HNRNPF, and promotes the expression of rG4-containing pre-mRNAs (the "rG4ome"). We observe an upregulation of the rG4ome during epithelial-to-mesenchymal transition and a negative correlation of rG4 abundance with patient survival in different cancer types. By preventing the nuclear accumulation of PKM2, we could repress the rG4ome in triple-negative breast cancer cells and reduce migration and invasion of cancer cells in vitro and in xenograft mouse models. Our data suggest that the balance of folded and unfolded rG4s controlled by RBPs impacts gene expression during tumor progression., Competing Interests: Declaration of interests J.R. is on the scientific advisory board and has an equity interest in Pearl Bio. J.R. is a co-founder and has an equity interest in Kapis Biosciences., (Published by Elsevier Inc.)

Ribosome biogenesis is a highly regulated cellular process requiring a large cohort of accessory factors to ensure the accurate production of ribosomes. Dysregulation of ribosome biogenesis is associated with the development of various human diseases, including cancer. The Las1L-Nol9 endonuclease-kinase complex is essential for the cleavage of the rRNA internal transcribed spacer 2 (ITS2), the phosphorylation of the 5'-hydroxyl end of the resulting precursor, and, thus, the maturation of the 60S ribosome. However, how the Las1L-Nol9 complex is regulated in cells is unclear. In this study, we report that the nucleolar ubiquitin-specific protease USP36 is a novel regulator of the Las1L-Nol9 complex. USP36 interacts with both Las1L and Nol9 and regulates their stability via deubiquitination. Intriguingly, USP36 also mediates the SUMOylation of Las1L, mainly at lysine (K) 565. Mutating K565 to arginine (R) does not affect the levels of Las1L and the formation of the Las1L-Nol9 complex, but abolishes its function in ITS2 processing, as unlike wild-type Las1L, the K565R mutant failed to rescue the defects in the ITS2 processing induced by the knockdown of endogenous Las1L. These results suggest that USP36-mediated Las1L SUMOylation is critical for ITS2 processing and that USP36 plays a critical role in ribosome biogenesis by regulating the Las1L-Nol9 complex., Significance: This study identifies USP36 as a deubiquitinating and small ubiquitin-like modifier ligase dual-function enzyme to mediate Las1L deubiquitination and SUMOylation. Las1L SUMOylation at K565 plays a critical role in pre-rRNA ITS2 processing. Thus, our study reveals a novel downstream pathway for USP36-regulated ribosome biogenesis., (©2024 The Authors; Published by the American Association for Cancer Research.)

The PIWI-interacting RNA (piRNA) pathway guides the DNA methylation of young, active transposons during germline development in male mice 1 . piRNAs tether the PIWI protein MIWI2 (PIWIL4) to the nascent transposon transcript, resulting in DNA methylation through SPOCD1 (refs. 2-5 ). Transposon methylation requires great precision: every copy needs to be methylated but off-target methylation must be avoided. However, the underlying mechanisms that ensure this precision remain unknown. Here, we show that SPOCD1 interacts directly with SPIN1 (SPINDLIN1), a chromatin reader that primarily binds to H3K4me3-K9me3 (ref. 6 ). The prevailing assumption is that all the molecular events required for piRNA-directed DNA methylation occur after the engagement of MIWI2. We find that SPIN1 expression precedes that of both SPOCD1 and MIWI2. Furthermore, we demonstrate that young LINE1 copies, but not old ones, are marked by H3K4me3, H3K9me3 and SPIN1 before the initiation of piRNA-directed DNA methylation. We generated a Spocd1 separation-of-function allele in the mouse that encodes a SPOCD1 variant that no longer interacts with SPIN1. We found that the interaction between SPOCD1 and SPIN1 is essential for spermatogenesis and piRNA-directed DNA methylation of young LINE1 elements. We propose that piRNA-directed LINE1 DNA methylation requires a developmentally timed two-factor authentication process. The first authentication is the recruitment of SPIN1-SPOCD1 to the young LINE1 promoter, and the second is MIWI2 engagement with the nascent transcript. In summary, independent authentication events underpin the precision of piRNA-directed LINE1 DNA methylation., (© 2024. The Author(s).)

Competing Interests: Conflict of interest statement. None declared.

Alternative splicing (AS) plays important roles in modulating environmental stress responses in plants. However, little is known about the functions of bicarbonate-induced AS in cultivated soybean (Glycine max L. Merr.). In this study, we combined PacBio isoform sequencing (Iso-seq) and Illumina RNA sequencing (RNA-seq) to elucidate the bicarbonate-induced AS events in soybean root and leaf tissues. Compared to RNA-seq, Iso-seq identified more novel genes and transcripts, as well as more AS events, indicating that Iso-seq is more efficient in AS detection. Combining these two technologies, we found that intron retention (IR) is the most frequent AS event type. We identified a total of 913 and 1974 bicarbonate stress-responsive differentially alternative spliced genes (DAGs) in soybean leaves and roots respectively, from our RNA-seq results. Additionally, we determined a transcription factor (GmNTL9) and a splicing factor (GmRSZ22), and validated their roles in bicarbonate stress response by AS. Overall, our study opens an avenue for evaluating plant AS regulatory networks, and the obtained global landscape of alternative splicing provides valuable insights into the AS-mediated bicarbonate-responsive mechanisms in plant species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Proper regulation of mRNA production in the nucleus is critical for the maintenance of cellular homoeostasis during adaptation to internal and environmental cues. Over the past 25 years, it has become clear that the nuclear machineries governing gene transcription, pre-mRNA processing, pre-mRNA and mRNA decay, and mRNA export to the cytoplasm are inextricably linked to control the quality and quantity of mRNAs available for translation. More recently, an ever-expanding diversity of new mechanisms by which nuclear RNA decay factors finely tune the expression of protein-encoding genes have been uncovered. Here, we review the current understanding of how mammalian cells shape their protein-encoding potential by regulating the decay of pre-mRNAs and mRNAs in the nucleus., (© 2024. Springer Nature Limited.)

The alternative splicing of pre-mRNA generates distinct mRNA variants from a pre-mRNA, thereby modulating a gene's function. The splicing of pre-mRNA depends on splice sites and regulatory elements in pre-mRNA, as well as the snRNA and proteins that recognize these sequences. Among these, RNA-binding proteins (RBPs) are the primary regulators of pre-mRNA splicing and play a critical role in the regulation of alternative splicing by recognizing the elements in pre-mRNA. However, little is known about the function of RBPs in stress response in plants. Here, we summarized the RBPs involved in the alternative splicing of pre-mRNA and their recognizing elements in pre-mRNA, and the recent advance in the role of RBP-mediated alternative splicing in response to abiotic stresses in plants. This review proposes that the regulation of pre-mRNA alternative splicing by RBPs is an important way for plants to adapt to abiotic stresses, and the regulation of alternative splicing by RBPs is a promising direction for crop breeding.

As a reader of tri-methylated lysine 36 on histone H3 (H3K36me3), Npac has been shown to have a significant role in gene transcription elongation. However, its potential implication in RNA splicing remains unknown. Here, we characterized the phenotypes of Npac knockout in mES cells. We discovered that loss of Npac disrupts pluripotency and identity in mESCs. We also found that Npac is associated with many cellular activities, including cell proliferation, differentiation, and transcription regulation. Notably, we uncovered that Npac is associated with RNA splicing machinery. Furthermore, we found that Npac regulates alternative splicing through its interaction with the splicing factors, including Srsf1. Our research thus highlights the important role of Npac in maintaining ESC identity through the regulation of pre-mRNA splicing.

Pre-rRNA processing is essential to ribosome biosynthesis. However, the processing mechanism is not fully understood in plants. Here, we report a DEAD-box RNA helicase DEK51 that mediates the 3' end processing of 18S and 5.8S pre-rRNA in maize (Zea mays L.). DEK51 is localized in the nucleolus, and loss of DEK51 arrests maize seed development and blocks the 3' end processing of 18S and 5.8S pre-rRNA. DEK51 interacts with putative key factors in nuclear RNA exosome-mediated pre-rRNA processing, including ZmMTR4, ZmSMO4, ZmRRP44A, and ZmRRP6L2. This suggests that DEK51 facilitates pre-rRNA processing by interacting with the exosome. Loss of ZmMTR4 function arrests seed development and blocks the 3' end processing of 18S and 5.8S pre-rRNA, similar to dek51. DEK51 also interacts with endonucleases ZmUTP24 and ZmRCL1, suggesting that it may also be involved in the cleavage at site A2. These results show the critical role of DEK51 in promoting 3' end processing of pre-rRNA., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ribosomal RNAs are processed in a complex pathway. We profiled rRNA processing intermediates in yeast at single-molecule and single-nucleotide levels with circularization, targeted amplification and deep sequencing (CircTA-seq), gaining significant mechanistic insights into rRNA processing and surveillance. The long form of the 5' end of 5.8S rRNA is converted to the short form and represents an intermediate of a unified processing pathway. The initial 3' end processing of 5.8S rRNA involves trimming by Rex1 and Rex2 and Trf4-mediated polyadenylation. The 3' end of 25S rRNA is formed by sequential digestion by four Rex proteins. Intermediates with an extended A1 site are generated during 5' degradation of aberrant 18S rRNA precursors. We determined precise polyadenylation profiles for pre-rRNAs and show that the degradation efficiency of polyadenylated 20S pre-rRNA critically depends on poly(A) lengths and degradation intermediates released from the exosome are often extensively re-polyadenylated., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

CircRNA, an essential RNA molecule involved in various biological functions and diseases, often exhibits decreased expression in tumor tissues, playing a role as a tumor suppressor, and suggesting therapeutic potential for cancer. However, current methods for promoting circRNA production are limited. This study introduces a novel approach for enhancing circRNA biogenesis, termed circRNA promoting RNA (cpRNA). CpRNA is designed to complement the flanking sequences of reverse complementary matches (RCMs) within pre-mRNA, thereby facilitating circRNA formation through improved exon circularization. Using a split-GFP reporter system, we demonstrated that cpRNA significantly enhance circGFP production. Optimization identified the best conditions for cpRNA to promote circRNA biogenesis, and these cpRNAs were then used to augment the production of endogenous circRNAs. These results indicate that cpRNAs can specifically increase the production of endogenous circRNAs with RCMs, such as circZKSCAN1 and circSMARCA5 in cancer cells, thereby inhibiting cell proliferation and migration by modulating circRNA-related pathways, showcasing the therapeutic potential of cpRNAs. Mechanistic studies have also shown that cpRNA promotes circRNA biogenesis, in part, by antagonizing the unwinding function of DHX9. Overall, these findings suggest that cpRNA represents a promising strategy for circRNA overexpression, offering a potential treatment for diseases marked by low circRNA levels., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ribosomal RNAs are transcribed as part of larger precursor molecules. In Escherichia coli , complementary RNA segments flank each rRNA and form long leader-trailer (LT) helices, which are crucial for subunit biogenesis in the cell. A previous study of 15 representative species suggested that most but not all prokaryotes contain LT helices. Here, we use a combination of in silico folding and covariation methods to identify and characterize LT helices in 4464 bacterial and 260 archaeal organisms. Our results suggest that LT helices are present in all phyla, including Deinococcota, which had previously been suspected to lack LT helices. In very few organisms, our pipeline failed to detect LT helices for both 16S and 23S rRNA. However, a closer case-by-case look revealed that LT helices are indeed present but escaped initial detection. Over 3600 secondary structure models, many well supported by nucleotide covariation, were generated. These structures show a high degree of diversity. Yet, all exhibit extensive base-pairing between the leader and trailer strands, in line with a common and essential function., (© 2024 Gemler et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Many RNA-binding proteins (RBPs) are linked to the dysregulation of RNA metabolism in motor neuron diseases (MNDs). However, the molecular mechanisms underlying MN vulnerability have yet to be elucidated. Here, we found that such an RBP, Quaking5 (Qki5), contributes to formation of the MN-specific transcriptome profile, termed "MN-ness," through the posttranscriptional network and maintenance of the mature MNs. Immunohistochemical analysis and single-cell RNA sequencing (scRNA-seq) revealed that Qki5 is predominantly expressed in MNs, but not in other neuronal populations of the spinal cord. Furthermore, comprehensive RNA sequencing (RNA-seq) analyses revealed that Qki5-dependent RNA regulation plays a pivotal role in generating the MN-specific transcriptome through pre-messenger ribonucleic acid (mRNA) splicing for the synapse-related molecules and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling pathways. Indeed, MN-specific ablation of the Qki5 caused neurodegeneration in postnatal mice and loss of Qki5 function resulted in the aberrant activation of stress-responsive JNK/SAPK pathway both in vitro and in vivo. These data suggested that Qki5 plays a crucial biological role in RNA regulation and safeguarding of MNs and might be associated with pathogenesis of MNDs., Competing Interests: Competing interests statement:H.O. is a paid member of the Scientific Advisory Boards of San Bio Co., Ltd., and K Pharma Inc. M. Yano is a paid member of the Scientific Advisory Board of K Pharma Inc.

The ribosome small subunit (SSU) is assembled by the SSU processome which contains approximately 70 non-ribosomal protein factors. Whilst the biochemical mechanisms of the SSU processome in 18S rRNA processing and maturation have been extensively studied, how SSU processome components enter the nucleolus has yet to be systematically investigated. Here, in examining the nucleolar localization of 50 human SSU processome components, we found that UTP3, together with another 24 proteins, enter the nucleolus autonomously. For the remaining 25 proteins we found that UTP3/SAS10 assists the nucleolar localization of five proteins (MPP10, UTP25, EMG1 and the two UTP-B components UTP12 and UTP13), likely through its interaction with nuclear importin α. This 'ferrying' function of UTP3 was then confirmed as conserved in the zebrafish. We also found that knockdown of human UTP3 impairs cleavage at the A0-site while loss-of-function of either utp3/sas10 or utp13/tbl3 in zebrafish causes the accumulation of aberrantly processed 5'ETS products, which highlights the crucial role of UTP3 in mediating 5'ETS processing. Mechanistically, we found that UTP3 facilitates the degradation of processed 5'ETS by recruiting the RNA exosome component EXOSC10 to the nucleolus. These findings lay the groundwork for studying the mechanism of cytoplasm-to-nucleolus trafficking of SSU processome components., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Non-segmented negative-strand (NNS) RNA viruses, such as rabies, Nipah and Ebola, produce 5'-capped and 3'-polyadenylated mRNAs resembling higher eukaryotic mRNAs. Here, we developed a transcription elongation-coupled pre-mRNA capping system for vesicular stomatitis virus (VSV, a prototypic NNS RNA virus). Using this system, we demonstrate that the single-polypeptide RNA-dependent RNA polymerase (RdRp) large protein (L) catalyzes all pre-mRNA modifications co-transcriptionally in the following order: (i) 5'-capping (polyribonucleotidylation of GDP) to form a GpppA cap core structure, (ii) 2'-O-methylation of GpppA into GpppAm, (iii) guanine-N7-methylation of GpppAm into m7GpppAm (cap 1), (iv) 3'-polyadenylation to yield a poly(A) tail. The GDP polyribonucleotidyltransferase (PRNTase) domain of L generated capped pre-mRNAs of 18 nucleotides or longer via the formation of covalent enzyme-pre-mRNA intermediates. The single methyltransferase domain of L sequentially methylated the cap structure only when pre-mRNAs of 40 nucleotides or longer were associated with elongation complexes. These results suggest that the formation of pre-mRNA closed loop structures in elongation complexes via the RdRp and PRNTase domains followed by the RdRp and MTase domains on the same polypeptide is required for the cap 1 formation during transcription. Taken together, our findings indicate that NNS RNA virus L acts as an all-in-one viral mRNA assembly machinery., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Competing Interests: Conflict of interest statement. None declared.

Exportin5 (Exp5) is the major miRNA nuclear export factor and recognizes structural features of pre-miRNA hairpins, while it also exports other minihelix-containing RNAs. In Drosophila, Exp5 is suggested to play a major role in tRNA export because the gene encoding the canonical tRNA export factor Exportin-t is missing in its genome. To understand molecular functions of fly Exp5, we studied the Exp5/RNA interactome in the cell line S2R + using the crosslinking and immunoprecipitation (CLIP) technology. The CLIP experiment captured known substrates such as tRNAs and miRNAs and detected candidates of novel Exp5 substrates including various mRNAs and long non-coding RNAs (lncRNAs). Some mRNAs and lncRNAs enriched PAR-CLIP tags compared to their expression levels, suggesting selective binding of Exp5 to them. Intronless mRNAs tended to enrich PAR-CLIP tags; therefore, we proposed that Exp5 might play a role in the export of specific classes of mRNAs/lncRNAs. This result suggested that Drosophila Exp5 might have a wider variety of substrates than initially thought. Surprisingly, Exp5 CLIP reads often contained sequences corresponding to the flanking 5'-leaders and 3'-trailers of tRNAs, which were thought to be removed prior to nuclear export. In fact, we found pre-tRNAs before end-processing were present in the cytoplasm, supporting the idea that tRNA end-processing is a cytoplasmic event. In summary, our results provide a genome-wide list of Exp5 substrate candidates and suggest that flies may lack a mechanism to distinguish pre-tRNAs with or without the flanking sequences., Competing Interests: Conflicts of interest The authors declare that they have no conflcits of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Genotoxic stress, arising from various environmental sources and endogenous cellular processes, pose a constant threat to genomic stability. Cells have evolved intricate mechanisms to detect and repair DNA damage, orchestrating a robust genotoxic stress response to safeguard the integrity of the genome. Recent research has shed light on the crucial role of co- and post-transcriptional regulatory mechanisms in modulating the cellular response to genotoxic stress. Here we highlight recent advances illustrating the intricate interplay between pre-mRNA processing, with a focus on 3'-end processing, and genotoxic stress response., (© 2024 The Authors. BioEssays published by Wiley Periodicals LLC.)

The heterogeneity of CARM1 controls first cell fate bias during early mouse development. However, how this heterogeneity is established is unknown. Here, we show that Carm1 mRNA is of a variety of specific exon-skipping splicing (ESS) isoforms in mouse two-cell to four-cell embryos that contribute to CARM1 heterogeneity. Disruption of paraspeckles promotes the ESS of Carm1 precursor mRNAs (pre-mRNAs). LincGET, but not Neat1, is required for paraspeckle assembly and inhibits the ESS of Carm1 pre-mRNAs in mouse two-cell to four-cell embryos. We further find that LincGET recruits paraspeckles to the Carm1 gene locus through HNRNPU. Interestingly, PCBP1 binds the Carm1 pre-mRNAs and promotes its ESS in the absence of LincGET. Finally, we find that the ESS seen in mouse two-cell to four-cell embryos decreases CARM1 protein levels and leads to trophectoderm fate bias. Our findings demonstrate that alternative splicing of CARM1 has an important role in first cell fate determination., (© 2024. The Author(s).)

The formation of new ribosomes is tightly coordinated with cell growth and proliferation. In eukaryotes, the correct assembly of all ribosomal proteins and RNAs follows an intricate scheme of maturation and rearrangement steps across three cellular compartments: the nucleolus, nucleoplasm, and cytoplasm. We demonstrate that usnic acid, a lichen secondary metabolite, inhibits the maturation of the large ribosomal subunit in yeast. We combine biochemical characterization of pre-ribosomal particles with a quantitative single-particle cryo-EM approach to monitor changes in nucleolar particle populations upon drug treatment. Usnic acid rapidly blocks the transition from nucleolar state B to C of Nsa1-associated pre-ribosomes, depleting key maturation factors such as Dbp10 and hindering pre-rRNA processing. This primary nucleolar block rapidly rebounds on earlier stages of the pathway which highlights the regulatory linkages between different steps. In summary, we provide an in-depth characterization of the effect of usnic acid on ribosome biogenesis, which may have implications for its reported anti-cancer activities., (© 2024. The Author(s).)

The tumor suppressor p53 and its antagonists MDM2 and MDM4 integrate stress signaling. For instance, dysbalanced assembly of ribosomes in nucleoli induces p53. Here, we show that the ribosomal protein L22 (RPL22; eL22), under conditions of ribosomal and nucleolar stress, promotes the skipping of MDM4 exon 6. Upon L22 depletion, more full-length MDM4 is maintained, leading to diminished p53 activity and enhanced cellular proliferation. L22 binds to specific RNA elements within intron 6 of MDM4 that correspond to a stem-loop consensus, leading to exon 6 skipping. Targeted deletion of these intronic elements largely abolishes L22-mediated exon skipping and re-enables cell proliferation, despite nucleolar stress. L22 also governs alternative splicing of the L22L1 (RPL22L1) and UBAP2L mRNAs. Thus, L22 serves as a signaling intermediate that integrates different layers of gene expression. Defects in ribosome synthesis lead to specific alternative splicing, ultimately triggering p53-mediated transcription and arresting cell proliferation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

pre-mRNA splicing is a critical feature of eukaryotic gene expression. Both cis- and trans-splicing rely on accurately recognising splice site sequences by spliceosomal U snRNAs and associated proteins. Spliceosomal snRNAs carry multiple RNA modifications with the potential to affect different stages of pre-mRNA splicing. Here, we show that the conserved U6 snRNA m6A methyltransferase METT-10 is required for accurate and efficient cis- and trans-splicing of C. elegans pre-mRNAs. The absence of METT-10 in C. elegans and METTL16 in humans primarily leads to alternative splicing at 5' splice sites with an adenosine at +4 position. In addition, METT-10 is required for splicing of weak 3' cis- and trans-splice sites. We identified a significant overlap between METT-10 and the conserved splicing factor SNRNP27K in regulating 5' splice sites with +4A. Finally, we show that editing endogenous 5' splice site +4A positions to +4U restores splicing to wild-type positions in a mett-10 mutant background, supporting a direct role for U6 snRNA m6A modification in 5' splice site recognition. We conclude that the U6 snRNA m6A modification is important for accurate and efficient pre-mRNA splicing., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Acute liver failure (ALF) is characterized by the rapidly progressive deterioration of hepatic function, which, without effective medical intervention, results in high mortality and morbidity. Here, using proteomic and transcriptomic analyses in murine ALF models, we found that the expression of multiple splicing factors was downregulated in ALF. Notably, we found that KH-type splicing regulatory protein (KHSRP) has a protective effect in ALF. Knockdown of KHSRP resulted in dramatic splicing defects, such as intron retention, and led to the exacerbation of liver injury in ALF. Moreover, we demonstrated that KHSRP directly interacts with splicing factor 3b subunit 1 (SF3B1) and enhances the binding of SF3B1 to the intronic branch sites, thereby promoting pre-mRNA splicing. Using splicing inhibitors, we found that Khsrp protects against ALF by regulating pre-mRNA splicing in vivo. Overall, our findings demonstrate that KHSRP is an important splicing activator and promotes the expression of genes associated with ALF progression by interacting with SF3B1; thus, KHSRP could be a possible target for therapeutic intervention in ALF., (© 2024. The Author(s).)

The eukaryotic mRNA surveillance pathway, a pivotal guardian of mRNA fidelity, stands at the nexus of diverse biological processes, including antiviral immunity. Despite the recognized function of splicing factors on mRNA fate, the intricate interplay shaping the mRNA surveillance pathway remains elusive. We illustrate that the conserved splicing factor U2 snRNP auxiliary factor large subunit B (U2AF65B) modulates splicing of mRNA surveillance complex, contributing to transcriptomic homeostasis in maize. The functionality of the mRNA surveillance pathway requires ZmU2AF65B-mediated normal splicing of upstream frameshift 3 ( ZmUPF3 ) pre-mRNA, encoding a core factor in this pathway. Intriguingly, sugarcane mosaic virus (SCMV)-coded nuclear inclusion protein a protease (NIa-Pro) hinders the splicing function of ZmU2AF65B. Furthermore, NIa-Pro disrupts ZmU2AF65B binding to ZmUPF3 pre-mRNA, leading to dysregulated splicing of ZmUPF3 transcripts and, consequently, impairing mRNA surveillance, thus facilitating viral infection. Together, this study establishes that splicing governs the mRNA surveillance pathway and identifies a pathogenic protein capable of disrupting this regulation to compromise RNA immunity.

MicroRNAs (miRNAs) are essential regulators of gene expression, defined by their unique biogenesis, which requires the precise excision of the small RNA from an imperfect fold-back precursor. Unlike their animal counterparts, plant miRNA precursors exhibit variations in sizes and shapes. Plant MIRNAs can undergo processing in a base-to-loop or loop-to-base direction, with DICER-LIKE1 (DCL1) releasing the miRNA after two cuts (two-step MIRNAs) or more (sequential MIRNAs). In this study, we demonstrate the critical role of the miRNA/miRNA* duplex region in the processing of miRNA precursors. We observed that endogenous MIRNAs frequently experience suboptimal processing in vivo due to mismatches in the miRNA/miRNA* duplex, a key region that fine-tunes miRNA levels. Enhancing the interaction energy of the miRNA/miRNA* duplex in two-step MIRNAs results in a substantial increase in miRNA levels. Conversely, sequential MIRNAs display distinct and specific requirements for the miRNA/miRNA* duplexes along their foldback structure. Our work establishes a connection between the miRNA/miRNA* structure and precursor processing mechanisms. Furthermore, we reveal a link between the biological function of miRNAs and the processing mechanism of their precursors with the evolution of plant miRNA/miRNA* duplex structures., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The eukaryotic nucleus has a highly organized structure. Although the spatiotemporal arrangement of spliceosomes on nascent RNA drives splicing, the nuclear architecture that directly supports this process remains unclear. Here, we show that RNA-binding proteins (RBPs) assembled on RNA form meshworks in human and mouse cells. Core and accessory RBPs in RNA splicing make two distinct meshworks adjacently but distinctly distributed throughout the nucleus. This is achieved by mutual exclusion dynamics between the charged and uncharged intrinsically disordered regions (IDRs) of RBPs. These two types of meshworks compete for spatial occupancy on pre-mRNA to regulate splicing. Furthermore, the optogenetic enhancement of the RBP meshwork causes aberrant splicing, particularly of genes involved in neurodegeneration. Genetic mutations associated with neurodegenerative diseases are often found in the IDRs of RBPs, and cells harboring these mutations exhibit impaired meshwork formation. Our results uncovered the spatial organization of RBP networks to drive RNA splicing., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Drosophila RNA-binding protein (RBP) Nab2 acts in neurons to regulate neurodevelopment and is orthologous to the human intellectual disability-linked RBP, ZC3H14. Nab2 governs axon projection in mushroom body neurons and limits dendritic arborization of class IV sensory neurons in part by regulating splicing events in ∼150 mRNAs. Analysis of the Sex-lethal ( Sxl ) mRNA revealed that Nab2 promotes an exon-skipping event and regulates m 6 A methylation on Sxl pre-mRNA by the Mettl3 methyltransferase. Mettl3 heterozygosity broadly rescues Nab2 null phenotypes implying that Nab2 acts through similar mechanisms on other RNAs, including unidentified targets involved in neurodevelopment. Here, we show that Nab2 and Mettl3 regulate the removal of a 5'UTR (untranslated region) intron in the trio pre-mRNA. Trio utilizes two GEF domains to balance Rac and RhoGTPase activity. Intriguingly, an isoform of Trio containing only the RhoGEF domain, GEF2, is depleted in Nab2 null nervous tissue. Expression of Trio-GEF2 rescues projection defects in Nab2 null axons and dendrites, while the GEF1 Rac1-regulatory domain exacerbates these defects, suggesting Nab2-mediated regulation Trio-GEF activities. Collectively, these data indicate that Nab2-regulated processing of trio is critical for balancing Trio-GEF1 and -GEF2 activity and show that Nab2, Mettl3, and Trio function in a common pathway that shapes axon and dendrite morphology., Competing Interests: Conflict of interest: The authors declare no financial conflict of interest.

In eukaryotes, pre-mRNA splicing is vital for RNA processing and orchestrated by the spliceosome, whose assembly starts with the interaction between U1-70K and SR proteins. Despite the significance of the U1-70K/SR interaction, the dynamic nature of the complex and the challenges in obtaining soluble U1-70K have impeded a comprehensive understanding of the interaction at the structural level for decades. We overcome the U1-70K solubility issues, enabling us to characterize the interaction between U1-70K and SRSF1, a representative SR protein. We unveil specific interactions: phosphorylated SRSF1 RS with U1-70K BAD1, and SRSF1 RRM1 with U1-70K RRM. The RS/BAD1 interaction plays a dominant role, whereas the interaction between the RRM domains further enhances the stability of the U1-70K/SRSF1 complex. The RRM interaction involves the C-terminal extension of U1-70K RRM and the conserved acid patches on SRSF1 RRM1 that is involved in SRSF1 phase separation. Our circular dichroism spectra reveal that BAD1 adapts an α-helical conformation and RS is intrinsically disordered. Intriguingly, BAD1 undergoes a conformation switch from α-helix to β-strand and random coil upon RS binding. In addition to the regulatory mechanism via SRSF1 phosphorylation, the U1-70K/SRSF1 interaction is also regulated by U1-70K BAD1 phosphorylation. We find that U1-70K phosphorylation inhibits the U1-70K and SRSF1 interaction. Our structural findings are validated through in vitro splicing assays and in-cell saturated domain scanning using the CRISPR method, providing new insights into the intricate regulatory mechanisms of pre-mRNA splicing., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

A universal characteristic of eukaryotic transcription is that the promoter recruits RNA polymerase II (RNAPII) to produce both precursor mRNAs (pre-mRNAs) and short unstable promoter upstream transcripts (PROMPTs) toward the opposite direction. However, how the transcription machinery selects the correct direction to produce pre-mRNAs is largely unknown. Here, through multiple acute auxin-inducible degradation systems, we show that rapid depletion of an RNAPII-binding protein complex, Integrator, results in robust PROMPT accumulation throughout the genome. Interestingly, the accumulation of PROMPTs is compensated by the reduction of pre-mRNA transcripts in actively transcribed genes. Consistently, Integrator depletion alters the distribution of polymerase between the sense and antisense directions, which is marked by increased RNAPII-carboxy-terminal domain Tyr1 phosphorylation at PROMPT regions and a reduced Ser2 phosphorylation level at transcription start sites. Mechanistically, the endonuclease activity of Integrator is critical to suppress PROMPT production. Furthermore, our data indicate that the presence of U1 binding sites on nascent transcripts could counteract the cleavage activity of Integrator. In this process, the absence of robust U1 signal at most PROMPTs allows Integrator to suppress the antisense transcription and shift the transcriptional balance in favor of the sense direction., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Precursor-mRNA (pre-mRNA) splicing requires the assembly, remodelling and disassembly of the multi-megadalton ribonucleoprotein complex called the spliceosome 1 . Recent studies have shed light on spliceosome assembly and remodelling for catalysis 2-6 , but the mechanism of disassembly remains unclear. Here we report cryo-electron microscopy structures of nematode and human terminal intron lariat spliceosomes along with biochemical and genetic data. Our results uncover how four disassembly factors and the conserved RNA helicase DHX15 initiate spliceosome disassembly. The disassembly factors probe large inner and outer spliceosome surfaces to detect the release of ligated mRNA. Two of these factors, TFIP11 and C19L1, and three general spliceosome subunits, SYF1, SYF2 and SDE2, then dock and activate DHX15 on the catalytic U6 snRNA to initiate disassembly. U6 therefore controls both the start 5 and end of pre-mRNA splicing. Taken together, our results explain the molecular basis of the initiation of canonical spliceosome disassembly and provide a framework to understand general spliceosomal RNA helicase control and the discard of aberrant spliceosomes., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

N 6 -methyladenosine (m 6 A) is the most abundant modification to mRNAs. Loss-of-function studies of main m 6 A regulators have indicated the role of m 6 A in pre-mRNA splicing. Recent studies have reported the role of splicing in preventing m 6 A deposition. Understanding the interplay between m 6 A and mRNA splicing holds the potential to clarify the significance of these fundamental molecular mechanisms in cell development and function, thereby shedding light on their involvement in the pathogenesis of myriad diseases., Competing Interests: Declaration of Competing Interest There is no competing interest related to the submitted manuscript., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Alternative polyadenylation (APA) is a crucial mechanism for regulating gene expression during pre-mRNA 3' processing. Pre-mRNA 3' end processing factors is the main factor involved in this process. However, pre-mRNA 3' end processing factors in different cancer expression profiles and the relationship between pre-mRNA 3' end processing factors and tumor microenvironment and the prognosis of the same patient is still unclear. In this study, we conducted a comprehensive exploration of the core pre-mRNA 3' end processing factors across various cancer types by utilizing common cancer database, and revealing a robust correlation between the expression of these core factors and tumor characteristics. Leveraging advanced bioinformatics databases, we evaluated the expression levels and prognostic relevance of pre-mRNA 3' end processing factors across pan-cancer tissues. Our extensive pan-cancer analysis revealed unique expression patterns of pre-mRNA 3' end processing factors in both tumor and adjacent non-tumorous tissues. Notably, we found a significant correlation between the expression levels of pre-mRNA 3' end processing factors and patient prognosis. Furthermore, we identified strong associations between pre-mRNA 3' end processing factors expression and various factors, such as stromal, immune, RNA stemness, and DNA stemness scores across pan-cancer tissues. Our data also highlighted a link between the expression of pre-mRNA 3' end processing factors and sensitivity to specific drugs, including pyrazoloacndine, amonaflide, and chelerythrinede, among others. We found four key pre-mRNA 3' end processing factors that play a crucial role in mRNA preprocessing. Our study illuminates the potential promotion and inhibition role of pre-mRNA 3' end processing regulators in the progression of cancer, CPSF2, CPSF3, CSTF2, SYMPK offering valuable insights for future research investigations on these regulators as diagnostic markers and therapeutic targets across pan-cancer., (© 2024. The Author(s).)

The spliceosome executes pre-mRNA splicing through four sequential stages: assembly, activation, catalysis, and disassembly. Activation of the spliceosome, namely remodeling of the pre-catalytic spliceosome (B complex) into the activated spliceosome (B act complex) and the catalytically activated spliceosome (B * complex), involves major flux of protein components and structural rearrangements. Relying on a splicing inhibitor, we have captured six intermediate states between the B and B * complexes: pre-B act , B act -I, B act -II, B act -III, B act -IV, and post-B act . Their cryo-EM structures, together with an improved structure of the catalytic step I spliceosome (C complex), reveal how the catalytic center matures around the internal stem loop of U6 snRNA, how the branch site approaches 5'-splice site, how the RNA helicase PRP2 rearranges to bind pre-mRNA, and how U2 snRNP undergoes remarkable movement to facilitate activation. We identify a previously unrecognized key role of PRP2 in spliceosome activation. Our study recapitulates a molecular choreography of the human spliceosome during its catalytic activation., (© 2024. The Author(s).)

The twenty-three Fanconi anemia (FA) proteins cooperate in the FA/BRCA pathway to repair DNA interstrand cross-links (ICLs). The cell division cycle and apoptosis regulator 1 (CCAR1) protein is also a regulator of ICL repair, though its possible function in the FA/BRCA pathway remains unknown. Here, we demonstrate that CCAR1 plays a unique upstream role in the FA/BRCA pathway and is required for FANCA protein expression in human cells. Interestingly, CCAR1 co-immunoprecipitates with FANCA pre-mRNA and is required for FANCA mRNA processing. Loss of CCAR1 results in retention of a poison exon in the FANCA transcript, thereby leading to reduced FANCA protein expression. A unique domain of CCAR1, the EF hand domain, is required for interaction with the U2AF heterodimer of the spliceosome and for excision of the poison exon. Taken together, CCAR1 is a splicing modulator required for normal splicing of the FANCA mRNA and other mRNAs involved in various cellular pathways., Competing Interests: Declaration of interests A.D.D. reports consulting for AbbVie, Deerfield Management Company, Impact Therapeutics, Moderna Therapeutics, PrimeFour Therapeutics, Schrödinger Inc., Servier BioInnovation LLC, and Tango Therapeutics; is a Scientific Advisory Board Member and Stockholder for Impact Therapeutics and Covant Therapeutics. K.A. is a member of the Advisory Board of Molecular Cell, the SAB of CAMP4 Therapeutics, consults for Syros Pharmaceuticals and Odyssey Therapeutics, and received research funding from Novartis not related to this work., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

3'Untranslated regions (3'UTRs) are essential portions of genes containing elements necessary for pre-mRNA 3'end processing and are involved in post-transcriptional gene regulation. Despite their importance, they remain poorly characterized in eukaryotes. Here, we have used a multi-pronged approach to extract and curate 3'UTR data from 11533 publicly available datasets, corresponding to the entire collection of Caenorhabditis elegans transcriptomes stored in the NCBI repository from 2009 to 2023. We have also performed high throughput cloning pipelines to identify and validate rare 3'UTR isoforms and incorporated and manually curated 3'UTR isoforms from previously published datasets. This updated C. elegans 3'UTRome (v3) is the most comprehensive resource in any metazoan to date, covering 97.4% of the 20362 experimentally validated protein-coding genes with refined and updated 3'UTR boundaries for 23489 3'UTR isoforms. We also used this novel dataset to identify and characterize sequence elements involved in pre-mRNA 3'end processing and update miRNA target predictions. This resource provides important insights into the 3'UTR formation, function, and regulation in eukaryotes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: The progression of non-small cell lung cancer (NSCLC) is significantly influenced by circular RNAs (circRNAs), especially in tumor hypoxia microenvironment. However, the precise functions and underlying mechanisms of dysregulated circRNAs in NSCLC remain largely unexplored., Methods: Differentially expressed circRNAs in NSCLC tissues were identified through high-throughput RNA sequencing. The characteristics of circ_0007386 were rigorously confirmed via Sanger sequencing, RNase R treatment and actinomycin D treatment. The effects of circ_0007386 on proliferation and apoptosis were investigated using CCK8, cloning formation assays, TUNEL staining, and flow cytometry assays in vitro. In vivo, xenograft tumor models were used to evaluate its impact on proliferation. Mechanistically, the regulatory relationships of circ_0007386, miR-383-5p and CIRBP were examined through dual luciferase reporter assays and rescue experiments. Additionally, we detected the binding of EIF4A3 to CRIM1 pre-mRNA by RNA immunoprecipitation and the interaction between YAP1 and EIF4A3 under hypoxic conditions by co-immunoprecipitation., Results: Our investigation revealed a novel circRNA, designated as circ_0007386, that was upregulated in NSCLC tissues and cell lines. Circ_0007386 modulated proliferation and apoptosis in NSCLC both in vitro and in vivo. Functionally, circ_0007386 acted as a sponge for miR-383-5p, targeting CIRBP, which influenced NSCLC cell proliferation and apoptosis via the PI3K/AKT signaling pathway. Furthermore, under hypoxic conditions, the interaction between YAP1 and EIF4A3 was enhanced, leading to the displacement of EIF4A4 from binding to CRIM1 pre-mRNA. This facilitated the back-splicing of CRIM1 pre-mRNA, increasing the formation of circ_0007386. The circ_0007386/miR-383-5p/CIRBP axis was significantly associated with the clinical features and prognosis of NSCLC patients., Conclusions: Circ_0007386, regulated by YAP1-EIF4A3 interaction under hypoxia conditions, plays an oncogenic role in NSCLC progression via the miR-383-5p/CIRBP axis., (© 2024. The Author(s).)

Identification of splice sites is a critical step in pre-messenger RNA (pre-mRNA) splicing because the definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 small nuclear ribonucleoprotein (snRNP). This involves both base-pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5' splice site (5'ss)/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3), which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger (ZnF) domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. Although we were unable to determine a function for the first ZnF domain, humanization of the second ZnF domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5'ss. In contrast, the corresponding ZnF domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5'ss. Our work reveals a role for the second ZnF of Luc7 in splice site selection and suggests that different ZnF domains may have different ATPase requirements for release by Prp28., (© 2024 Carrocci et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The recognition of the 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand, the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 (yU1) snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yU1 snRNA and the 5' ss RNA duplex. We replaced the zinc-finger (ZnF) domain of yeast U1C (yU1C) with its human counterpart, which resulted in a cold-sensitive growth phenotype and moderate splicing defects. We next added an auxin-inducible degron to yeast Luc7 (yLuc7) protein (to mimic the lack of Luc7Ls in human U1 snRNP). We found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of Prp40 and Snu71 (two other essential yU1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe 2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yU1 snRNP, and splicing plays a major role in the regulation of mitochondrial function in yeast., (© 2024 Chalivendra et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Mirtrons represent a subclass of microRNAs (miRNAs) that rely on the splicing machinery for their maturation. However, the molecular details of this Drosha-independent processing are still not fully understood; as an example, the Microprocessor complex cannot process the mirtronic pre-miRNA from the transcript even if splice site mutations are present. To investigate the influence of alternative splicing sites on mirtron formation, we generated Enhanced Green Fluorescent Protein (EGFP) reporters containing artificial introns to compare the processing of canonical miRNAs and mirtrons. Although mutations of both splice sites generated a complex pattern of alternative transcripts, mirtron formation was always severely affected as opposed to the normal processing of the canonical hsa-mir-33b miRNA. However, we also detected that while its formation was also hindered, the mirtron-derived hsa-mir-877-3p miRNA was less affected by certain mutations than the hsa-mir-877-5p species. By knocking down Drosha, we showed that this phenomenon is not dependent on Microprocessor activity but rather points toward the potential stability difference between the miRNAs from the different arms. Our results indicate that when the major splice sites are mutated, mirtron formation cannot be rescued by nearby alternative splice sites, and stability differences between 5p and 3p species should also be considered for functional studies of mirtrons.

Acute myeloid leukemia (AML) is frequently linked to genetic abnormalities, with the t (8; 21) translocation, resulting in the production of a fusion oncoprotein AML1-ETO (AE), being a prevalent occurrence. This protein plays a pivotal role in t (8; 21) AML's onset, advancement, and recurrence, making it a therapeutic target. However, the development of drug molecules targeting AML1-ETO are markedly insufficient, especially used in clinical treatment. In this study, it was uncovered that Neratinib could significantly downregulate AML1-ETO protein level, subsequently promoting differentiation of t (8; 21) AML cells. Based on "differentiated active" probes, Neratinib was identified as a functional inhibitor against HNRNPA3 through covalent binding. The further studies demonstrated that HNRNPA3 function as a putative m6A reader responsible for recognizing and regulating the alternative splicing of AML-ETO pre-mRNA. These findings not only contribute to a novel insight to the mechanism governing post-transcriptional modification of AML1-ETO transcript, but also suggest that Neratinib would be promising therapeutic potential for t (8; 21) AML treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pre-mRNA splicing is a significant step for post-transcriptional modifications and functions in a wide range of physiological processes in plants. Human NHP2L binds to U4 snRNA during spliceosome assembly; it is involved in RNA splicing and mediates the development of human tumors. However, no ortholog has yet been identified in plants. Therefore, we report At4g12600 encoding the ortholog NHP2L protein, and AtSNU13 associates with the component of the spliceosome complex; the atsnu13 mutant showed compromised resistance in disease resistance, indicating that AtSNU13 is a positive regulator of plant immunity. Compared to wild-type plants, the atsnu13 mutation resulted in altered splicing patterns for defense-related genes and decreased expression of defense-related genes, such as RBOHD and ALD1. Further investigation shows that AtSNU13 promotes the interaction between U4/U6.U5 tri-snRNP-specific 27 K and the motif in target mRNAs to regulate the RNA splicing. Our study highlights the role of AtSNU13 in regulating plant immunity by affecting the pre-mRNA splicing of defense-related genes., (© 2024. The Author(s).)

Pre-messenger RNA (pre-mRNA) splicing modulation is an attractive approach for investigating the mechanisms of genetic disorders caused by mis-splicing. Previous reports have indicated that a modified U7 small nuclear RNA (U7 snRNA) is a prospective tool for modulating splicing both in vitro and in vivo. To date, very few studies have investigated the role of antisense sequence length in modified U7 snRNA. In this study, we designed a series of antisense sequences with various lengths and evaluated their efficiency in inducing splicing modulation. To express modified U7 snRNAs, we constructed a series of plasmid DNA sequences which codes cytomegalovirus (CMV) enhancer, human U1 promoter, and modified mouse U7 snRNAs with antisense sequences of different lengths. We evaluated in vitro splicing modulation efficiency using a luciferase reporter system for simple and precise evaluation as well as reverse transcription-polymerase chain reaction to monitor splicing patterns. Our in vitro assay findings suggest that antisense sequences of modified mouse U7 snRNAs have an optimal length for efficient splicing modulation, which depends on the target exon. In addition, antisense sequences that were either too long or too short decreased splicing modulation efficiency. To confirm reproducibility, we performed an in vitro assay using two target genes, mouse Fas and mouse Dmd. Together, our data suggests that the antisense sequence length should be optimized for modified mouse U7 snRNAs to induce efficient splicing modulation., Competing Interests: Authors are employees of Chugai Pharmaceutical Co., Ltd., (Copyright: © 2024 Shimo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)