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Main conclusion: Serine/arginine-rich (SR) proteins participate in RNA processing by interacting with precursor mRNAs or other splicing factors to maintain plant growth and stress responses. Alternative splicing is an important mechanism involved in mRNA processing and regulation of gene expression at the posttranscriptional level, which is the main reason for the diversity of genes and proteins. The process of alternative splicing requires the participation of many specific splicing factors. The SR protein family is a splicing factor in eukaryotes. The vast majority of SR proteins’ existence is an essential survival factor. Through its RS domain and other unique domains, SR proteins can interact with specific sequences of precursor mRNA or other splicing factors and cooperate to complete the correct selection of splicing sites or promote the formation of spliceosomes. They play essential roles in the composition and alternative splicing of precursor mRNAs, providing pivotal functions to maintain growth and stress responses in animals and plants. Although SR proteins have been identified in plants for three decades, their evolutionary trajectory, molecular function, and regulatory network remain largely unknown compared to their animal counterparts. This article reviews the current understanding of this gene family in eukaryotes and proposes potential key research priorities for future functional studies. [ABSTRACT FROM AUTHOR]

Purpose: Hereditary angioedema (HAE) is a rare autosomal dominant life-threatening disease characterized by low levels of C1 inhibitor (type I HAE) or normal levels of ineffective C1 inhibitor (type II HAE), typically occurring as a consequence of a SERPING1 mutation. In some cases, a causal mutation remains undetected after using a standard molecular genetic analysis. Results: Here we show a long methodological way to the final discovery of c.1029 + 384A > G, a novel deep intronic mutation in intron 6 which is responsible for HAE type I in a large family and has not been identified by a conventional diagnostic approach. This mutation results in de novo donor splice site creation and subsequent pseudoexon inclusion, the mechanism firstly described to occur in SERPING1 in this study. We additionally discovered that the proximal part of intron 6 is a region potentially prone to pseudoexon-activating mutations, since natural alternative exons and additional cryptic sites occur therein. Indeed, we confirmed the existence of at least two different alternative exons in this region not described previously. Conclusions: In conclusion, our results suggest that detecting aberrant transcripts, which are often low abundant because of nonsense-mediated decay, requires a modified methodological approach. We suggest SERPING1 intron 6 sequencing and/or tailored mRNA analysis to be routinely used in HAE patients with no mutation identified in the coding sequence. [ABSTRACT FROM AUTHOR]

Key message: Functions of most splice isoforms that are generated by alternative splicing are unknown. We show that two splice variants that encode proteins differing in only eight amino acids have distinct functions in a stress response. Serine/arginine-rich (SR) and SR-like proteins, a conserved family of RNA binding proteins across eukaryotes, play important roles in pre-mRNA splicing and other post-transcriptional processes. Pre-mRNAs of SR and SR-like proteins undergo extensive alternative splicing in response to diverse stresses and produce multiple splice isoforms. However, the functions of most splice isoforms remain elusive. Alternative splicing of pre-mRNA of Arabidopsis SR45, which encodes an SR-like splicing regulator, generates two isoforms (long—SR45.1 and short—SR45.2). The proteins encoded by these two isoforms differ in eight amino acids. Here, we investigated the role of SR45 and its splice variants in salt stress tolerance. The loss of SR45 resulted in enhanced sensitivity to salt stress and changes in expression and splicing of genes involved in regulating salt stress response. Interestingly, only the long isoform (SR45.1) rescued the salt-sensitive phenotype as well as the altered gene expression and splicing patterns in the mutant. These results suggest that SR45 positively regulates salt tolerance. Furthermore, only the long isoform is required for SR45-mediated salt tolerance. [ABSTRACT FROM AUTHOR]

Pre-mRNA splicing is an essential step in gene expression, when introns are removed and exons joined by the complex of proteins called spliceosome. Correct splicing requires a precise exon/intron junction definition, which is determined by a consensual donor and acceptor splice site at the 5′ and 3′ end, respectively. An acceptor splice site (3′ss) consists of highly conserved AG nucleotides in positions E−2 and E−1. These nucleotides can appear in tandem, located 3 bp from each other. Then they are referred to as NAGNAG or tandem 3′ss, which can be alternatively spliced. NAG/TAG 3′ss motif abundance is extremely low and cannot be easily explained by just a nucleotide preference in this position. We tested artificial NAG/TAG motif's potential negative effect on exon recognition using a minigene assay. Introducing the NAG/TAG motif into seven different exons revealed no general negative effect on exon recognition. The only observed effect was the partial use of the newly formed distal 3′ss. We can conclude that this motif's extremely low preference in a natural 3′ss is not a consequence of the NAG/TAG motif's negative effect on exon recognition, but more likely the result of other RNA processing aspects, such as an alternative 3′ss choice, decreased 3′ss strength, or incorporating an amber stop codon. [ABSTRACT FROM AUTHOR]

The spliceosome is necessary for the removal of intron sequences in transcribed pre-mRNA in eukaryotic cells. More than 200 proteins are involved in the process of pre-mRNA splicing; they coordinate the alternative splicing of most eukaryotic genes, which participate in the regulation of a variety of important cellular processes. However, a growing number of studies demonstrate the functions of spliceosomal proteins that are not directly related to the splicing process, such as (1) R-loop formation, (2) DNA repair, (3) telomere elongation, (4) M phase regulation, and (5) mRNA export from the nucleus. In this review, we summarize the data on the noncanonical functions of the spliceosomal proteins. Taking into account the fact that mutations in spliceosomal genes are commonly found in tumor cells, a careful study of noncanonical functions of spliceosomal proteins may provide new insights into the development of effective methods of cancer treatment and design of drugs for delaying cell aging. [ABSTRACT FROM AUTHOR]

Key message: Molecular and functional characterization of four gene families of the Physalis exon junction complex (EJC) core improved our understanding of the evolution and function of EJC core genes in plants.Abstract: The exon junction complex (EJC) plays significant roles in posttranscriptional regulation of genes in eukaryotes. However, its developmental roles in plants are poorly known. We characterized four EJC core genes from Physalis floridana that were named PFMAGO, PFY14, PFeIF4AIII and PFBTZ. They shared a similar phylogenetic topology and were expressed in all examined organs. PFMAGO, PFY14 and PFeIF4AIII were localized in both the nucleus and cytoplasm while PFBTZ was mainly localized in the cytoplasm. No protein homodimerization was observed, but they could form heterodimers excluding the PFY14-PFBTZ heterodimerization. Virus-induced gene silencing (VIGS) of PFMAGO or PFY14 aborted pollen development and resulted in low plant survival due to a leaf-blight-like phenotype in the shoot apex. Carpel functionality was also impaired in the PFY14 knockdowns, whereas pollen maturation was uniquely affected in PFBTZ-VIGS plants. Once PFeIF4AIII was strongly downregulated, plant survival was reduced via a decomposing root collar after flowering and Chinese lantern morphology was distorted. The expression of Physalis orthologous genes in the DYT1-TDF1-AMS-bHLH91 regulatory cascade that is associated with pollen maturation was significantly downregulated in PFMAGO-, PFY14- and PFBTZ-VIGS flowers. Intron-retention in the transcripts of P. floridana dysfunctional tapetum1 (PFDYT1) occurred in these mutated flowers. Additionally, the expression level of WRKY genes in defense-related pathways in the shoot apex of PFMAGO- or PFY14-VIGS plants and in the root collar of PFeIF4AIII-VIGS plants was significantly downregulated. Taken together, the Physalis EJC core genes play multiple roles including a conserved role in male fertility and newly discovered roles in Chinese lantern development, carpel functionality and defense-related processes. These data increase our understanding of the evolution and functions of EJC core genes in plants. [ABSTRACT FROM AUTHOR]

Precise pre-mRNA splicing, essential for appropriate protein translation, depends on the presence of consensus “cis” sequences that define exon-intron boundaries and regulatory sequences recognized by splicing machinery. Point mutations at these consensus sequences can cause improper exon and intron recognition and may result in the formation of an aberrant transcript of the mutated gene. The splicing mutation may occur in both introns and exons and disrupt existing splice sites or splicing regulatory sequences (intronic and exonic splicing silencers and enhancers), create new ones, or activate the cryptic ones. Usually such mutations result in errors during the splicing process and may lead to improper intron removal and thus cause alterations of the open reading frame. Recent research has underlined the abundance and importance of splicing mutations in the etiology of inherited diseases. The application of modern techniques allowed to identify synonymous and nonsynonymous variants as well as deep intronic mutations that affected pre-mRNA splicing. The bioinformatic algorithms can be applied as a tool to assess the possible effect of the identified changes. However, it should be underlined that the results of such tests are only predictive, and the exact effect of the specific mutation should be verified in functional studies. This article summarizes the current knowledge about the “splicing mutations” and methods that help to identify such changes in clinical diagnosis. [ABSTRACT FROM AUTHOR]

The dim1+ gene family is essential for G2/M transition during mitosis and encodes a small nuclear ribonucleoprotein that functions in the mRNA splicing machinery of eukaryotes. However, the plant homolog of DIM1 gene has not been defined yet. Here, we identified a gene named GmDim1 positioned on chromosome 9 of soybean (Glycine max (L.) Merr.) with 80% homology to other eukaryotic dim1+ family genes. A domain of soybean DIM1 protein was primarily conserved with U5 snRNP protein family and secondarily aligned with mitotic DIM1 protein family. The GmDim1 gene was expressed constitutively in all soybean organs. The transgenic Arabidopsis thaliana (L.) plants overexpressing GmDim1 showed early flowering and stem elongation, produced multiple shoots and continued flowering after the post-flowering stage. DIM1 proteins transiently expressed in onion cells were localized in the nucleus with dense deposition in the nucleolus. Therefore, we propose that the soybean GmDim1 gene is a component of plant U5 snRNP involved in mRNA splicing and normal progress of plant growth. [ABSTRACT FROM AUTHOR]

Pre-mRNA splicing is an essential component of eukaryotic gene expression. Many metazoans, including humans, regulate alternative splicing patterns to generate expansions of their proteome from a limited number of genes. Importantly, a considerable fraction of human disease causing mutations manifest themselves through altering the sequences that shape the splicing patterns of genes. Thus, understanding the mechanistic bases of this complex pathway will be an essential component of combating these diseases. Dating almost to the initial discovery of splicing, researchers have taken advantage of the genetic tractability of budding yeast to identify the components and decipher the mechanisms of splicing. However, budding yeast lacks the complex splicing machinery and alternative splicing patterns most relevant to humans. More recently, many researchers have turned their efforts to study the fission yeast, Schizosaccharomyces pombe, which has retained many features of complex splicing, including degenerate splice site sequences, the usage of exonic splicing enhancers, and SR proteins. Here, we review recent work using fission yeast genetics to examine pre-mRNA splicing, highlighting its promise for modeling the complex splicing seen in higher eukaryotes. [ABSTRACT FROM AUTHOR]

Transient receptor potential (TRP) channels are unusual among cation channels because of their diverse cation selectivities and activation mechanisms. TRP channels thus play major roles in various sensory perceptions by functioning as multimodal signal integrators. Some TRP subfamily members are also implicated in acute and chronic pain and inflammation. So far, most TRP channel studies have been targeted to human and model organisms within a limited evolutionary context. Classification of TRP channels in various animal genomes has revealed extensive gene gain and loss events across animal species. Furthermore, the chemical activation profiles of some orthologous TRP channels were different between species such as human and mouse. Amino acid substitutions must underlie such differences, and the crucial amino acid residues have been identified in some cases. These changes represent the evolution of TRP channels at the amino acid sequence level. There is also evidence that TRP channels have obtained species-diversity through alternative splicing and possibly cis-regulatory element mutations. All of the above demonstrate the dynamic and plastic evolutionary history of metazoan TRP channels at multiple levels, possibly in conjunction with the specific habitats and life histories of individual species. [ABSTRACT FROM AUTHOR]

The characteristic high regenerative ability of plants has been exploited to develop in vitro plant regeneration techniques, which are usually initiated by an in vitro dedifferentiation step induced by artificial phytohormone treatment. Recent advances in plant molecular biological and genetic technologies have revealed the importance of the regulation of RNA metabolism, including the control of rRNA biosynthesis, pre-mRNA splicing, and miRNA-based RNA decay, in successful in vitro dedifferentiation. This review provides a brief overview of current knowledge of the roles of RNA metabolism in the dedifferentiation of plant cells in vitro. In addition, the possibility that plant-specific aspects of RNA metabolism regulation are linked closely to their high regenerative ability is discussed. [ABSTRACT FROM AUTHOR]

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). Deficiency of BTK leads to a developmental block in B cell differentiation; hence, the patients essentially lack antibody-producing plasma cells and are susceptible to various infections. A substantial portion of the mutations in BTK results in splicing defects, consequently preventing the formation of protein-coding mRNA. Antisense oligonucleotides (ASOs) are therapeutic compounds that have the ability to modulate pre-mRNA splicing and alter gene expression. The potential of ASOs has been exploited for a few severe diseases, both in pre-clinical and clinical studies. Recently, advances have also been made in using ASOs as a personalized therapy for XLA. Splice-correction of BTK has been shown to be feasible for different mutations in vitro, and a recent proof-of-concept study demonstrated the feasibility of correcting splicing and restoring BTK both ex vivo and in vivo in a humanized bacterial artificial chromosome (BAC)-transgenic mouse model. This review summarizes the advances in splice correction, as a personalized medicine for XLA, and outlines the promises and challenges of using this technology as a curative long-term treatment option. [ABSTRACT FROM AUTHOR]

The mammalian genome harbors thousands of long noncoding RNA (lncRNA) genes. Recent studies have indicated the involvement of several of these lncRNAs in the regulation of gene expression. lncRNAs play crucial roles in various biological processes ranging from epigenetic gene regulation, transcriptional control, to post-transcriptional regulation. lncRNAs are localized in various subcellular compartments, and major proportion of these are retained in the cell nucleus and could be broadly classified as nuclear-retained lncRNAs (nrRNAs). Based on the identified functions, members of the nrRNAs execute diverse roles, including providing architectural support to the hierarchical subnuclear organization and influencing the recruitment of chromatin modifier factors to specific chromatin sites. In this review, we will summarize the recently described roles of mammalian nrRNAs in controlling gene expression by influencing chromatin organization, transcription, pre-mRNA processing, nuclear organization, and their involvement in disease. [ABSTRACT FROM AUTHOR]

Alternative splicing (AS) regulation of pre-mRNA has been proven to be one of the fundamental layers of plant immune system. How pathogens disrupt plant AS process to suppress plant immunity by secreted effectors remain poorly understood. In the recent study, Gui et al. revealed that a previously identified effector PSR1 of Phytophthora interferes with host RNA splicing machinery to modulate small RNA biogenesis, leading to compromised plant immunity. The study provided a novel insight into the importance of AS process during pathogen-host interactions., (© 2022. The Author(s).)

Alternative splicing is a major source of protein diversity in humans. The human splicing factor zinc finger, Ran-binding domain containing protein 2 (ZRANB2) is a splicing protein whose specific endogenous targets are unknown. Its upregulation in grade III ovarian serous papillary carcinoma could suggest a role in some cancers. To determine whether ZRANB2 is part of the supraspliceosome, nuclear supernatants from human embryonic kidney 293 cells were prepared and then fractioned on a glycerol gradient, followed by Western blotting. The same was done after treatment with a tyrosine kinase to induce phosphorylation. This showed for the first time that ZRANB2 is part of the supraspliceosome, and that phosphorylation affects its subcellular location. Studies were then performed to understand the splicing targets of ZRANB2 at the whole-transcriptome level. HeLa cells were transfected with a vector containing ZRANB2 or with a vector-only control. RNA was extracted, converted to cDNA and hybridized to Affymetrix GeneChip Human Exon 1.0 ST Arrays. At the FDR ≤1.3 significance level we found that ZRANB2 influenced the alternative splicing of primary transcripts of CENTB1, WDR78, C10orf18, CABP4, SMARCC2, SPATA13, OR4C6, ZNF263, CAPN10, SALL1, ST18 and ZP2. Several of these have been implicated in tumor development. In conclusion ZRANB2 is part of the supraspliceosome and causes differential splicing of numerous primary transcripts, some of which might have a role in cancer. [ABSTRACT FROM AUTHOR]

Fas is a transmembrane cell surface protein recognized by Fas ligand (FasL). When FasL binds to Fas, the target cells undergo apoptosis. A soluble Fas molecule that lacks the transmembrane domain is produced from skipping of exon 6 encoding this region in alternative splicing procedure. The soluble Fas molecule has the opposite function of intact Fas molecule, protecting cells from apoptosis. Here we show that knockdown of hnRNP A1 promotes exon 6 skipping of Fas pre-mRNA, whereas overexpression of hnRNP A1 reduces exon 6 skipping. Based on the bioinformatics approach, we have hypothesized that hnRNP A1 functions through interrupting 5′ splice site selection of exon 5 by interacting with its potential binding site close to 5′ splice site of exon 5. Consistent with our hypothesis, we demonstrate that mutations of the hnRNP A1 binding site on exon 5 disrupted the effects of hnRNP A1 on exon 6 inclusion. RNA pull-down assay and then western blot analysis with hnRNP A1 antibody prove that hnRNP A1 contacts the potential binding site RNA sequence on exon 5 but not the mutant sequence. In addition, we show that the mutation of 5′ splice site on exon 5 to a less conserved sequence destructed the effects of hnRNP A1 on exon 6 inclusion. Therefore we conclude that hnRNP A1 interacts with exon 5 to promote distal exon 6 inclusion of Fas pre-mRNA. Our study reveals a novel alternative splicing mechanism of Fas pre-mRNA. [ABSTRACT FROM AUTHOR]

In recent years, an increasing number of mutations in what would appear to be 'housekeeping genes' have been identified as having unexpectedly specific defects in multicellular organogenesis. This is also the case for organogenesis in seed plants. Although it is not surprising that loss-of-function mutations in 'housekeeping' genes result in lethality or growth retardation, it is surprising when (1) the mutant phenotype results from the loss of function of a 'housekeeping' gene and (2) the mutant phenotype is specific. In this review, by defining housekeeping genes as those encoding proteins that work in basic metabolic and cellular functions, we discuss unexpected links between housekeeping genes and specific developmental processes. In a surprising number of cases housekeeping genes coding for enzymes or proteins with functions in basic cellular processes such as transcription, post-transcriptional modification, and translation affect plant development. [ABSTRACT FROM AUTHOR]

The hnRNP A/B proteins are among the most abundant RNA-binding proteins, forming the core of the ribonucleoprotein complex that associates with nascent transcripts in eukaryotic cells. There are several paralogs in this subfamily, each of which is subject to alternative transcript splicing and post-translational modifications. The structural diversity of these proteins generates a multitude of functions that involve interactions with DNA or, more commonly, RNA. They also recruit regulatory proteins associated with pathways related to DNA and RNA metabolism, and appear to accompany transcripts throughout the life of the mRNA. We have highlighted here recent progress in elucidation of molecular mechanisms underlying the roles of these hnRNPs in a wide range of nuclear processes, including DNA replication and repair, telomere maintenance, transcription, pre-mRNA splicing, and mRNA nucleo-cytoplasmic export. [ABSTRACT FROM AUTHOR]

In eukaryotes, most protein-coding genes contain introns which are removed by precursor messenger RNA (pre-mRNA) splicing. Alternative splicing is a process by which multiple messenger RNAs (mRNAs) are generated from a single pre-mRNA, resulting in functionally distinct proteins. Recent genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene expression. Mis-regulation of splicing causes a wide range of human diseases. This review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA splicing. It also discusses emerging directions in the field of alternative splicing. [ABSTRACT FROM AUTHOR]

Spatial organization of splicing machinery elements in metabolically and functionally different pollen cells during Hyacinthus orientalis pollen grain development was examined by localization of trimethylguanosine (TMG) snRNA and Sm proteins, representing splicing small nuclear ribonucleoproteins, as well as SR splicing factors was investigated. In young pollen grains the level of all labeled antigens was the highest displaying essentially uniform distribution in the vegetative and generative nucleus. In the polarized microspore, as well as in the vegetative cell of the young pollen grain, both TMG snRNA and Sm proteins were also found highly concentrated in Cajal bodies. After detachment of the generative cell from the sporoderm, the redistribution of splicing machinery elements into speckled-shape clusters was observed in both nuclei in the pollen. In the mature pollen grain, labeled antigens were still present, both in the vegetative and the generative nucleus. The results reflected that in differentiating H. orientalis pollen cells, the intracellular organization pre-mRNA splicing machinery undergoes significant and characteristic changes during the course of pollen grain development. Changes in the distribution of spliceosomal components relate to the transcriptional activity of both pollen cells during their maturation. [ABSTRACT FROM AUTHOR]

PurposeThis paper will illustrate how variation in the processing of mutant pre-mRNA can affect the phenotypic outcome of inherited disorders of type II collagen.MethodsType 1 Stickler's syndrome is one of the different phenotypes resulting from mutations in COL2A1 (the type II collagenopathies). It is also the commonest, but often goes undiagnosed due to the variability of phenotypic features, which in some cases may consist of only abnormal vitreous development. Most cases of type 1 Stickler's syndrome are due to premature termination codons in the mRNA, resulting in haploinsufficiency. This leaves a conundrum as to why the disease is so variable. Using RT-PCR of illegitimate transcript and also minigenes, we have investigated how certain mutations can variably affect mRNA processing.ResultsHere, we demonstrate and discuss how apparently similar mutations can have a dramatically different effect on splicing of the pre-mRNA, switching transcripts from ones which would be degraded by nonsense-mediated decay into messages that will be translated into mutant proteins that can exert a dominant-negative effect and ultimately modify the resulting phenotype.ConclusionVariability of Stickler's syndrome can, in part, be due to the variable effect that mutations have on the processing of the COL2A1 transcript.Eye (2008) 22, 1243–1250; doi:10.1038/eye.2008.34; published online 29 February 2008 [ABSTRACT FROM AUTHOR]

Mutations in the voltage-gated chloride/proton antiporter ClC-5 gene, CLCN5, are associated with Dent’s disease, an X-linked renal tubulopathy. Our interest is to identify and characterize disease-causing CLCN5 mutations, especially those that alter the splicing of the pre-mRNA. We analyzed the CLCN5 gene from nine unrelated Spanish Dent’s disease patients and their relatives by DNA sequencing. Pre-mRNA splicing analysis was performed by RT-PCR. Seven new mutations were identified, consisting of three missense mutations (C219R, F273L, and W547G), one splice-site mutation (IVS-2A > G), one deletion (976delG), and two non-sense mutations (Y140X and W314X). We found that missense mutation W547G also led to increased expression of a new alternative isoform lacking exons 10 and 11 that was expressed in several human tissues. In addition, we describe another novel CLCN5 splicing variant lacking exon 11 alone, which was expressed only in human skeletal muscle. We conclude that missense mutation W547G can also alter the expression levels of a CLCN5 mRNA splicing variant. This type of mutation has not been previously described in the CLCN5 gene. Our results support the importance of a routine analysis at the pre-mRNA level of mutations that are commonly assumed to cause single amino acids alterations. [ABSTRACT FROM AUTHOR]

We describe a fast and unambiguous method for haplotyping the (TG)mTn repeat in IVS8 and determining three other single nucleotide polymorphisms (SNPs) in exons 10, 14a and 24 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene affecting correct splicing of the CFTR pre-mRNA using primer extension and mass spectrometry. The diagnostic products are generated by primer extension (PEX) reactions, which require a single detection primer complementary to a region downstream of a target strand's variable site. On addition of a polymerase and an appropriate mixture of dNTP's and 2′, 3′-dideoxynucleotide triphosphates (ddNTP's), the primer is extended through the mutation region until the first ddNTP is incorporated and the mass of the extension products determines the composition of the variable site. Analysis of patient DNA assigned the correct and unambiguous haplotype for the (TG)mTn repeat in intron 8 of the CFTR gene. Additional crucial SNPs influencing correct splicing in exon 10, 14 and 24 can easily be detected by biplexing the assay to genotype allelic variants important for correct splicing of the CFTR pre-mRNA. Different PEX reactions with subsequent mass spectrometry generate sufficient data, to enable unambiguous and easy haplotyping of the (TG)mTn repeat in the CFTR gene. The method can be easily extended to the inclusion of additional SNPs of interest by biplexing some of the PEX reactions. All experimental steps required for PEX are amenable to the high degree of automation desirable for a high-throughput diagnostic setting, facilitating the work of clinicians involved in the diagnosis of non-classic cystic fibrosis.European Journal of Human Genetics (2007) 15, 53–61. doi:10.1038/sj.ejhg.5201725; published online 4 October 2006 [ABSTRACT FROM AUTHOR]

Heart failure results from the cumulative death of cardiomyocytes, and the inability of remaining cells to regenerate. Efforts toward transcriptional reprogramming of cardiomyocytes by overexpressing E1A or E2F1 have been limited by the inability of cardiomyocytes to enter and complete mitosis. Human CDC5 (hCDC5), a component of the pre-mRNA splicing complex, has been shown to regulate G2/M transit in asynchronously dividing cells. We now show that co-infection of recombinant adenoviruses expressing E1A/E1B and hCDC5 promotes cell cycle re-entry and G2/M progression in post-mitotic cardiomyocytes. Co-expression of E1A/E1B and hCDC5 induced nuclear localization of cyclin-dependent kinase 1 and cyclin B1, and was sufficient to promote mitotic entry as determined by an increase in mitotic index only in co-infected cells. E1A/E1B and hCDC5 promoted cell division, as evidenced by an increase in the number of cardiomyocytes following co-infection. Thus, overexpression of E1A/E1B and hCDC5 resulted in cell cycle re-entry, DNA synthesis, cell division, and an increase in cardiomyocyte number, suggesting the formation of new cardiomyocytes. These studies suggest that G1/S-phase transcriptional regulators, in combination with pre-mRNA splicing factors, such as CDC5, that regulate rate-limiting G2/M target genes may prove useful in developing therapies to stimulate myocardial regeneration.Gene Therapy (2006) 13, 837–843. doi:10.1038/sj.gt.3302737; published online 16 February 2006 [ABSTRACT FROM AUTHOR]

The spliceosomal component Prp1 (U5-102 kD) is found in Schizosaccharomyces pombe, a physiological substrate of Prp4 kinase. Here, we identify, spp41-1, a previously isolated extragenic suppressor of Prp4 kinase. The gene encodes an ATP-dependent RNA helicase homologous to the splicing factor Brr2 of Saccharomyces cerevisiae and U5-200 kD of mammalia. The suppressor allele, spp41-1, interacts genetically with alleles of prp1. We show that Prp1 and Brr2 are complexed in vivo with spliceosomal particles containing the five snRNAs U1, U2, U5, and base-paired U4/U6. Prp1 was found exclusively in small ribonucleoprotein particle (snRNP) complexes sedimenting in the range of 30S–60S, whereas Brr2 was also found sedimenting lower than 30S and free of snRNAs. Moreover, we find that the splicing factor Prp31 is complexed with Prp1 in the same spliceosomal particles containing the five snRNAs. These data indicate that in fission yeast spliceosomal particles larger than 30S exist, which can be considered as pre-catalytic spliceosomes. In addition, we show that S. pombe cells lacking Prp1 still contain these large pre-catalytic spliceosomal particles associated with Prp31. These data are consistent with the notion that in fission yeast phosphorylation of Prp1 by Prp4 kinase is involved in the activation of pre-catalytic spliceosomes. [ABSTRACT FROM AUTHOR]

The objective of the present study is to establish a minigene model for studying pre-mRNA alternative splicing. To prepare the minigene DNA constructs, with human or mouse genomic DNA as templates, GluR-B, FGF-2R and Zis “minigene” fragments were amplified using PCR and cloned to the eukaryotic expression vectors. The three constructed minigenes and the expression vectors of Tra2β1 and Zis2 were co-transfected in Hela cells. RT-PCR analysis was performed to semi-quantitatively determine the spliced products from the minigenes. The results demonstrated that the constructed minigenes are useful in studying the pre-mRNA alternative splicing in cultured cells. With the established Zis minigene, we for the first time found that Zis2 isoform regulates the alternative splicing of Zis minigene. [ABSTRACT FROM AUTHOR]

Most primary messenger RNA transcripts (pre-mRNAs) in eukaryotes contain intervening sequences that must be precisely removed to generate a functional mRNA. The excision of the intervening sequences, the introns, from a pre-mRNA and the concomitant joining of the flanking sequences, the exons, is called pre-mRNA splicing. Pre-mRNA splicing takes place in large ribonucleoprotein machinery, the spliceosome. Although the function and components of this machinery appear to be highly conserved between organisms, many distinct differences between budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe, have been found, emphasizing their evolutionary distance. Most interestingly, fission yeast appears to reflect the more conservative evolutionary development regarding pre-mRNA splicing. Many spliceosomal components, including the five small nuclear RNAs, which most likely form the catalytic core of the spliceosome, show a higher degree of similarity with the components of the splicing machinery found in mammals. In addition, several regulatory components of the spliceosome detected in mammals are absent in Sac. cerevisiae, but present in Sch. pombe. Here, we review recent progress made in our understanding of the control of pre-mRNA splicing in Sch. pombe. The focus is on Prp4p kinase, first discovered in fission yeast and also present in mammals, but absent in Sac. cerevisiae. Results from both mammals and Sch. pombe suggest that Prp4p plays a key role in regulating pre-mRNA splicing and in connecting this process with the cell cycle. [ABSTRACT FROM AUTHOR]

Nuclear extracts (NE), capable of carrying out splicing of pre-mRNA, contain galectin-1 and galectin-3. NE depleted of galectins-1 and -3 concomitantly lose their splicing activity. The activity of the galectin-depleted extract can be reconstituted by the addition of either galectin-1 or galectin-3. These results suggest that galectins-1 and -3 serve as redundant splicing factors. Consistent with this notion, immunofluorescence staining showed that both galectins yielded a diffuse nucleoplasmic distribution, matching that of nascent transcripts and consistent with the hypothesis that bulk transcription and pre-mRNA processing occur throughout the nucleoplasm. Under some conditions, the galectins could be found in speckled structures and nuclear bodies but the prevailing thought is that these represent sites of storage and recycling rather than sites of action. Galectin-1 and galectin-3 bind directly to Gemin4, a component of the SMN core complex, which plays multiple roles in ribonucleoprotein assembly, including the biogenesis, delivery, and recycling of snRNPs to the spliceosome. Thus, galectin-1 and galectin-3 constitute a part of an interacting dynamic network of many factors involved in the splicing and transport of mRNA. Published in 2004. [ABSTRACT FROM AUTHOR]

The structure of the gene encoding the apoprotein of phytochrome B1 (PHYB1) in tomato has been determined from genomic and cDNA sequences. In contrast to PHYA, PHYB1 lacks an intron upstream of the first ATG. A single transcription start site was found by 5′ RACE at –116. Tomato PHYB1 spans 7 kb starting from the first ATG. The coding region is organized into four exons as for other angiosperm PHY. The deduced apoprotein consists of 1131 amino acids, with a molecular mass of 125.4 kDa. Tomato phytochrome B1 shares 78% and 74% identity with Arabidopsis phytochromes B and D, respectively. Along with the normally spliced full-length transcripts, sequences of reverse transcriptase-PCR clones revealed five types of alternative transcripts. Each type of alternative transcript was missing a considerable part of the coding region, including the chromophore-binding site. The four putative PHYB1 mutants in tomato, which are temporarily red-light insensitive (tri), were each confirmed to have a mutation in PHYB1. Each mutation arose from a different, single-base substitution. Allele tri1 is presumably a null because the mutation introduces a stop at codon 92. In tri3, val-238 is replaced by Phe. The importance of this valine residue is evidenced by the fact that the tri3 phenotype is as strong as that of tri1. Alleles tri2 and tri4 encode proteins truncated at their C-termini. The former lacks either 170 or 438 amino acids, depending upon which of two types of splicing occurs during transcript maturation, while the latter lacks 225. [ABSTRACT FROM AUTHOR]

We have analysed 998 Arabidopsis intron sequences in the EMBL database. All Arabidopsis introns to adhere to the :GU...AG: rule with the exception of 1% of introns with :GC at their 5′ ends. Virtually all of the introns contained a putative branchpoint sequence (YUNAN) 18 to 60 nt upstream of the 3′ splice site. Although a polypyrimidine tract was much less apparent than in vertebrate introns, the most common nucleotide in the region upstream of the 3′ splice site was uridine. Consensus sequences for 5′ and 3′ splice sites and branchpoint sequences for Arabidopsis introns are presented. [ABSTRACT FROM AUTHOR]

The removal of introns from pre-mRNA transcripts and the concomitant ligation of exons is known as pre-mRNA splicing. It is a fundamental aspect of constitutive eukaryotic gene expression and an important level at which gene expression is regulated. The process is governed by multiple cis-acting elements of limited sequence content and particular spatial constraints, and is executed by a dynamic ribonucleoprotein complex termed the spliceosome. The mechanism and regulation of pre-mRNA splicing, and the sub-nuclear organisation of the spliceosomal machinery in higher plants is reviewed here. Heterologous introns are often not processed in higher plants indicating that, although highly conserved, the process of pre-mRNA splicing in plants exhibits significant differences that distinguish it from splicing in yeast and mammals. A fundamental distinguishing feature is the presence of and requirement for AU or U-rich intron sequence in higher-plant pre-mRNA splicing. In this review we document the properties of higher-plant introns and trans-acting spliceosomal components and discuss the means by which these elements combine to determine the accuracy and efficiency of pre-mRNA processing. We also detail examples of how introns can effect regulated gene expression by affecting the nature and abundance of mRNA in plants and list the effects of environmental stresses on splicing. Spliceosomal components exhibit a distinct pattern of organisation in higher-plant nuclei. Effective probes that reveal this pattern have only recently become available, but the domains in which spliceosomal components concentrate were identified in plant nuclei as enigmatic structures some sixty years ago. The organisation of spliceosomal components in plant nuclei is reviewed and these recent observations are unified with previous cytochemical and ultrastructural studies of plant ribonucleoprotein domains. [ABSTRACT FROM AUTHOR]

Phytochrome is the red/far-red absorbing photoreceptor active in photomorphogenesis, the apoprotein of which is encoded by a small gene family ( PHYA, PHYB, PHYC, PHYD and PHYE). A novel phytochrome B-deficient mutant, phyB-103, was isolated from a screen of EMS-mutagenised Arabidopsis M2 seed. phyB-103 carries a G-to-A base substitution at the 5′ splice site +1 G nucleotide of intron 1 of PHYB. The phyB-103 PHYB transcript is larger than the wild-type PHYB transcript and DNA sequence analysis showed that the entire intron is retained in the mature PHYB transcript of phyB-103. Thus the phyB-103 G-to-A substitution prevents intron splicing. The retained intron contains within it an in-frame stop codon, and the predicted PHYB-103 apoprotein thus terminates prematurely. phyB-103 is therefore likely to be a null allele of PHYB, consistent with the observation that the phenotype conferred by phyB-103 is as severe as that conferred by previously described phyB null alleles. [ABSTRACT FROM AUTHOR]

Our previous phylogenetic analysis based on cDNA sequences of chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPDH; genes GapA and GapC, respectively) of the red alga Chondrus crispus suggested that rhodophytes and green plants are sister groups with respect to plastids and mitochondria and diverged at about the same time or somewhat later than animals and fungi. Here we characterize the genomic sequences of genes GapC and GapA of C. crispus with respect to promotor structures, intron/exon organization, genomic complexity, G+C content, CpG suppression and their transcript levels in gametophytes and protoplasts, respectively. To our knowledge this is the first report on nuclear protein genes of red algae. The GapC gene is G+C-rich, contains no introns and displays a number of classic sequence motifs within its promotor region, such as TATA, CAAT, GC boxes and several elements resembling the plant-specific G-box palindrome. The GapA gene has a moderate G+C content, a single CAAT box motif in its promotor region and a single intron of 115 bp near its 5′ end. This intron occupies a conserved position corresponding to that of intron 1 in the transit peptide region of chloroplast GAPDH genes ( GapA and GapB) of higher plants. It has consensus sequences similar to those of yeast introns and folds into a conspicuous secondary structure of - 61.3 kJ. CpG profiles of genes GapC and GapA and their flanking sequences show no significant CpG depletion suggesting that these genomic sequences are not methylated. Genomic Southern blots hybridized with generic and gene specific probes indicate that both genes are encoded by single loci composed of multiple polymorphic alleles. Northern hybridizations demonstrate that both genes are expressed in gametophytes but not in protoplasts where appreciable amounts of transcripts can only be detected for GapC. [ABSTRACT FROM AUTHOR]

The small nuclear ribonucleoprotein particles U1, U2, U4/U6 and U5 participate in the removal of introns from pre-messenger RNAs in the nucleus. Three genes encoding U5snRNAs, the RNA moiety of U5snRNPs, have been isolated from maize. As in other plant UsnRNA gene families the three maize U5snRNA genes exhibit sequence variation. Two of the gene variants (MzU5.1 and MzU5.2) are clearly expressed after transfection into maize leaf protoplasts while the third gene variant (MzU5.3) is expressed at very low levels. These different levels of expression cannot be directly correlated with sequence changes in the highly conserved Upstream Sequence Element (USE) required for expression of Arabidopsis UsnRNA genes nor with differential stability of the U5snRNA transcripts. Further sequence elements may therefore have a role in regulating maize UsnRNA gene expression. [ABSTRACT FROM AUTHOR]

For successful splicing in dicot plants the only recognised intron requirements are 5′ and 3′ splice sites and AU-rich sequences. We have investigated further the importance of AU-rich elements by analyzing the splicing of an AU-rich antisense intron sequence. Activation of cryptic splice sites on either side of the AU-rich sequence permitted the efficient removal of this essentially non-intron sequence by splicing. This splicing event not only confirms the importance of AU-rich sequences but also has implications for the evolution of interrupted genes and the expression of heterologous genes in transgenic plants. [ABSTRACT FROM AUTHOR]

U1 small nuclear RNAs (U1snRNAs) occur in the nucleus of plants and animals where, complexed with several proteins in the form of U1 small nuclear ribonucleoprotein particles (U1snRNPs), they play an important role in precursor messenger RNA (pre-mRNA) splicing. Ten potato U1snRNA genes have been isolated on two genomic clones illustrating the clustering of this multigene family on the potato genome. Based on both the sequence of their coding regions and upstream regulatory elements, seven of the genes are potentially functional. The other three genes were pseudogenes with defective promoter or coding region sequences. Analysis of expression of individual cloned U1snRNA genes in transfected tobacco protoplasts was impossible due to the similarity of U1snRNA sequences in tobacco. However, by marking the coding regions with oligonucleotides or constructing chimaeric genes consisting of a potato U1snRNA promoter region and maize U5snRNA coding region, three of the U1 promoter regions were shown to be transcriptionally active. [ABSTRACT FROM AUTHOR]

We have isolated six new pre-mRNA splicing mutants ( prp) from a collection of temperature-sensitive (ts–) Schizosaccharomyces pombe strains. The prp mutants are defective in the splicing of both messenger RNA and U6 small nuclear RNA precursors. A single recessive mutation is responsible for both the ts – growth and the splicing phenotypes in each of the prp mutants. The six prp mutations are unlinked and fall into separate complementation groups. Two are allelic with the previously described prp3 and prp4 mutations; the remaining four define the new alleles prp5-1, prp6-1, prp7-1, and prp9-1. The six mutants exhibit three splicing phenotypes: accumulation of unspliced precursor at the restrictive but not at the permissive temperature; accumulation of unspliced precursor at both the permissive and restrictive temperatures; and accumulation of unspliced precursor, the intron-exon lariat intermediate, and the intron lariat final product. In addition to their aberrant splicing phenotypes, the prp5-1 and prp6-1 mutants express classical cell-division-cycle defects, while prp7-1 exhibits an unusual hyphal morphology. These results suggest a connection between pre-mRNA splicing and the control of cell division in fission yeast. [ABSTRACT FROM AUTHOR]

Snu13p is a highly conserved RNA binding protein from Saccharomyces cerevisiae required for both eukaryotic pre-mRNA splicing and pre-rRNA processing. The 1H, 13C, and 15N assignments were determined from multidimensional, multinuclear NMR experiments conducted at 25°C. [ABSTRACT FROM AUTHOR]