Interaction between FLASH and Lsm11 is essential for histone pre-mRNA processing in vivo in Drosophila

Metazoan replication-dependent histone mRNAs are the only nonpolyadenylated cellular mRNAs. Formation of the histone mRNA 3' end requires the U7 snRNP, which contains Lsm10 and Lsm11, and FLASH, a processing factor that binds Lsm11. Here, we identify sequences in Drosophila FLASH (dFLASH) that bind Drosophila Lsm11 (dLsm11), allow localization of dFLASH to the nucleus and histone locus body (HLB), and participate in histone pre-mRNA processing in vivo. Amino acids 105-154 of dFLASH bind to amino acids 1-78 of dLsm11. A two-amino acid mutation of dLsm11 that prevents dFLASH binding but does not affect localization of U7 snRNP to the HLB cannot rescue the lethality or histone pre-mRNA processing defects resulting from an Lsm11 null mutation. The last 45 amino acids of FLASH are required for efficient localization to the HLB in Drosophila cultured cells. Removing the first 64 amino acids of FLASH has no effect on processing in vivo. Removal of 13 additional amino acids of dFLASH results in a dominant negative protein that binds Lsm11 but inhibits processing of histone pre-mRNA in vivo. Inhibition requires the Lsm11 binding site, suggesting that the mutant dFLASH protein sequesters the U7 snRNP in an inactive complex and that residues between 64 and 77 of dFLASH interact with a factor required for processing. Together, these studies demonstrate that direct interaction between dFLASH and dLsm11 is essential for histone pre-mRNA processing in vivo and for proper development and viability in flies.     

PSF contacts exon 7 of SMN2 pre-mRNA to promote exon 7 inclusion

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease and a leading cause of infant mortality. Deletions or mutations of SMN1 cause SMA, a gene that encodes a SMN protein. SMN is important for the assembly of Sm proteins onto UsnRNA to UsnRNP. SMN has also been suggested to direct axonal transport of β-actin mRNA in neurons. Humans contain a second SMN gene called SMN2 thus SMA patients produce some SMN but not with sufficient levels. The majority of SMN2 mRNA does not include exon 7. Here we show that increased expression of PSF promotes inclusion of exon 7 in the SMN2 whereas reduced expression of PSF promotes exon 7 skipping. In addition, we present evidence showing that PSF interacts with the GAAGGA enhancer in exon 7. We also demonstrate that a mutation in this enhancer abolishes the effects of PSF on exon 7 splicing. Furthermore we show that the RNA target sequences of PSF and tra2β in exon 7 are partially overlapped. These results lead us to conclude that PSF interacts with an enhancer in exon 7 to promote exon 7 splicing of SMN2 pre-mRNA.     

Intercistronic region required for polycistronic pre-mRNA processing in Caenorhabditis elegans

In Caenorhabditis elegans, polycistronic pre-mRNAs are processed by cleavage and polyadenylation at the 3' ends of the upstream genes and trans splicing, generally to the specialized spliced leader SL2, at the 5' ends of the downstream genes. Previous studies have indicated a relationship between these two events in the processing of a heat shock-induced gpd-2-gpd-3 polycistronic pre-mRNA. Here, we report mutational analysis of the intercistronic region of this operon by linker scan analysis. Surprisingly, no sequences downstream of the 3' end were important for 3'-end formation. In contrast, a U-rich (Ur) element located 29 bp downstream of the site of 3'-end formation was shown to be important for downstream mRNA biosynthesis. This approximately 20-bp element is sufficient for SL2 trans splicing and mRNA accumulation when transplanted to a heterologous context. Furthermore, when the downstream gene was replaced by a gene from another organism, no loss of trans-splicing specificity was observed, suggesting that the Ur element may be the primary signal required for downstream mRNA processing.     

Modulation of alternative pre-mRNA splicing in vivo by pinin

Pre-mRNA splicing occurs in a large macromolecular RNA-protein complex called the spliceosome. The major components of the spliceosome include snRNP and SR proteins. We have previously identified an SR-like protein, pinin (pnn), which is localized not only in nuclear speckles but also at desmosomes. The nuclear localization of pnn is a dynamic process because pnn can be found not only with SR proteins in nuclear speckles but also in enlarged speckles following treatment of cells with RNA polymerase II inhibitors, DRB, and alpha-amanitin. Using adenovirus E1A and chimeric calcitonin/dhfr construct as a splicing reporter minigene in combination with cellular cotransfection, we found that pnn regulates alternative 5(') and 3(') splicing by decreasing the use of distal splice sites. Regulation of 5(') splice site choice was also observed for RNPS1, a general splicing activator that interacts with pnn in nuclear speckles. The regulatory ability of pnn in alternative 5(') splicing, however, was not dependent on RNPS1 and a pnn mutant, lacking the N-terminal 167 amino acids, behaved like a dominant negative species, inhibiting E1A splicing when applied in splicing assays. These results provide direct evidence that pnn functions as a splicing regulator which participates itself directly in splicing reaction or indirectly via other components of splicing machinery.     

LAMMER kinase Kic1 is involved in pre-mRNA processing

The LAMMER kinases are conserved through evolution. They play vital roles in cell growth/differentiation, development, and metabolism. One of the best known functions of the kinases in animal cells is the regulation of pre-mRNA splicing. Kic1 is the LAMMER kinase in fission yeast Schizosaccharomyces pombe. Despite the reported pleiotropic effects of kic1⁺ deletion/overexpression on various cellular processes the involvement of Kic1 in splicing remains elusive. In this study, we demonstrate for the first time that Kic1 not only is required for efficient splicing but also affects mRNA export, providing evidence for the conserved roles of LAMMER kinases in the unicellular context of fission yeast. Consistent with the hypothesis of its direct participation in multiple steps of pre-mRNA processing, Kic1 is predominantly present in the nucleus during interphase. In addition, the kinase activity of Kic1 plays a role in modulating its own cellular partitioning. Interestingly, Kic1 expression oscillates in a cell cycle-dependent manner and the peak level coincides with mitosis and cytokinesis, revealing a potential mechanism for controlling the kinase activity during the cell cycle. The novel information about the in vivo functions and regulation of Kic1 offers insights into the conserved biological roles fundamental to LAMMER kinases in eukaryotes.     

Splice site selection in polyomavirus late pre-mRNA processing.

Polyomavirus late pre-mRNAs contain one 5' splice site and two message body 3' splice sites, which are not used at equal frequencies. As a result of alternative splicing, the total late mRNA population consists of about 5% mVP2 (no message body splice chosen), about 15% mVP3 (promoter-proximal 3' splice site chosen), and about 80% mVP1 (promoter-distal 3' splice site chosen). To determine whether it is splice site strength that determines the ratio of spliced products, constructs containing duplicated or rearranged 3' splice sites were created. In construct VP1,1, 160 bp surrounding the VP3 3' splice site was substituted with the corresponding region of the VP1 3' splice site. This construct resulted in the duplication of the VP1 3' splicing signal. VP3,3 (two identical VP3 3' splice sites) and VP1,3 (VP1 and VP3 3' splice sites reversed) were similarly created. Each construct maintained wild-type spacing between the 3' splice sites. Analysis of RNAs from transfections showed that in each construct, the 3' splice closest to the polyadenylation site was used preferentially. Analysis of a number of additional constructs indicated that there are no strong cis-acting positive or negative regulators of polyomavirus late splicing; rather, splicing choices appear to be determined largely by relative position of splice sites.     

FRG1P-mediated aggregation of proteins involved in pre-mRNA processing

FRG1 is considered a candidate gene for facioscapulohumeral muscular dystrophy (FSHD) based on its location at chromosome 4qter and its upregulation in FSHD muscle. The FRG1 protein (FRG1P) localizes to nucleoli, Cajal bodies (and speckles), and has been suggested to be a component of the human spliceosome but its exact function is unknown. Recently, transgenic mice overexpressing high levels of FRG1P in skeletal muscle were described to present with muscular dystrophy. Moreover, upregulation of FRG1P was demonstrated to correlate with missplicing of specific pre-mRNAs. In this study, we have combined colocalization studies with yeast two-hybrid screens to identify proteins that associate with FRG1P. We demonstrate that artificially induced nucleolar aggregates of VSV-FRG1P specifically sequester proteins involved in pre-mRNA processing. In addition, we have identified SMN, PABPN1, and FAM71B, a novel speckle and Cajal body protein, as binding partners of FRG1P. All these proteins are, or seem to be, involved in RNA biogenesis. Our data confirm the presence of FRG1P in protein complexes containing human spliceosomes and support a potential role of FRG1P in either splicing or another step in nuclear RNA biogenesis. Intriguingly, among FRG1P-associated proteins are SMN and PABPN1, both being involved in neuromuscular disorders, possibly through RNA biogenesis-related processes.     

3' end processing of mouse histone pre-mRNA: evidence for additional base-pairing between U7 snRNA and pre-mRNA.

We have analysed the extent of base-pairing interactions between spacer sequences of histone pre-mRNA and U7 snRNA present in the trans-acting U7 snRNP and their importance for histone RNA 3' end processing in vitro. For the efficiently processed mouse H4-12 gene, a computer analysis revealed that additional base pairs could be formed with U7 RNA outside of the previously recognised spacer element (stem II). One complementarity (stem III) is located more 3' and involves nucleotides from the very 5' end of U7 RNA. The other, more 5' located complementarity (stem I) involves nucleotides of the Sm binding site of U7 RNA, a part known to interact with snRNP structural proteins. These potential stem structures are separated from each other by short internal loops of unpaired nucleotides. Mutational analyses of the pre-mRNA indicate that stems II and III are equally important for interaction with the U7 snRNP and for processing, whereas mutations in stem I have moderate effects on processing efficiency, but do not impair complex formation with the U7 snRNP. Thus nucleotides near the processing site may be important for processing, but do not contribute to the assembly of an active complex by forming a stem I structure. The importance of stem III was confirmed by the ability of a complementary mutation in U7 RNA to suppress a stem III mutation in a complementation assay using Xenopus laevis oocytes. The main role of the factor(s) binding to the upstream hairpin loop is to stabilise the U7-pre-mRNA complex. This was shown by either stabilising (by mutation) or destabilising (by increased temperature) the U7-pre-mRNA base-pairing under conditions where hairpin factor binding was either allowed or prevented (by mutation or competition). The hairpin dependence of processing was found to be inversely related to the strength of the U7-pre-mRNA interaction.     

Alternative trans-splicing: a novel mode of pre-mRNA processing

Alternative splicing is an important process contributing to proteome diversity without involving an increase in the number of genes. In some cases, alternative splicing is carried out under 'trans-mode', called alternative trans-splicing, in which exons located on separate pre-mRNA molecules are selectively joined to produce mature mRNAs encoding proteins with distinct structures and functions. However, it is not known how widespread or how frequently trans-splicing occurs in vivo. Recently, trans-allelic trans-splicing has been unambiguously demonstrated in Drosophila using a SNP (single nucleotide polymorphism) as a marker. In this review, we provide an overview of alternative trans-splicing in Drosophila and mammals, and discuss its mechanisms.