Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes in the yellow mealworm Tenebrio molitor

The nucleus of vitellogenic oocytes of the yellow mealworm Tenebrio molitor contains a karyosphere that consists of condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters (IGCs)) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged 2??-O-Me(U)₂₂ methyl oligoribonucleotide probes, complementary to poly(A) tails of RNAs, revealed poly(A)⁺ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein A1 localized to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and some nucleoplasmic IGCs also contain the adapter protein Aly known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was colocalized with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner, whereas it was RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data provide evidence for the implication of nucleoplasmic IGCs in mRNA biogenesis and retention on the path to nuclear export.     

RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA.     

Inhibition of splicing and nuclear retention of pre-mRNA by spliceostatin A in fission yeast

Nuclear retention of pre-mRNAs is tightly regulated by several security mechanisms that prevent pre-mRNA export into the cytoplasm. Recently, spliceostatin A, a methylated derivative of a potent antitumor microbial metabolite FR901464, was found to cause pre-mRNA accumulation and translation in mammalian cells. Here we report that spliceostatin A also inhibits splicing and nuclear retention of pre-mRNA in a fission yeast strain that lacks the multidrug resistance protein Pmd1. As observed in mammalian cells, spliceostatin A is bound to components of the SF3b complex in the spliceosome. Furthermore, overexpression of nup211, a homolog of Saccharomyces cerevisiae MLP1, suppresses translation of pre-mRNAs accumulated by spliceostatin A. These results suggest that the SF3b complex has a conserved role in pre-mRNA retention, which is independent of the Mlp1 function.     

RNA induces conformational changes in the SF1/U2AF65 splicing factor complex

Spliceosomes assemble on pre-mRNA splice sites through a series of dynamic ribonucleoprotein complexes, yet the nature of the conformational changes remains unclear. Splicing factor 1 (SF1) and U2 auxiliary factor (U2AF⁶⁵) cooperatively recognize the 3′ splice site during the initial stages of pre-mRNA splicing. Here, we used small-angle X-ray scattering to compare the molecular dimensions and ab initio shape restorations of SF1 and U2AF⁶⁵ splicing factors, as well as the SF1/U2AF⁶⁵ complex in the absence and presence of AdML (adenovirus major late) splice site RNAs. The molecular dimensions of the SF1/U2AF⁶⁵/RNA complex substantially contracted by 15 Å in the maximum dimension, relative to the SF1/U2AF⁶⁵ complex in the absence of RNA ligand. In contrast, no detectable changes were observed for the isolated SF1 and U2AF⁶⁵ splicing factors or their individual complexes with RNA, although slight differences in the shapes of their molecular envelopes were apparent. We propose that the conformational changes that are induced by assembly of the SF1/U2AF⁶⁵/RNA complex serve to position the pre-mRNA splice site optimally for subsequent stages of splicing.     

NSrp70 is a novel nuclear speckle-related protein that modulates alternative pre-mRNA splicing in vivo

Nuclear speckles are known to be the storage sites of mRNA splicing regulators. We report here the identification and characterization of a novel speckle protein, referred to as NSrp70, based on its subcellular localization and apparent molecular weight. This protein was first identified as CCDC55 by the National Institutes of Health Mammalian Gene Collection, although its function has not been assigned. NSrp70 was colocalized and physically interacted with SC35 and ASF/SF2 in speckles. NSrp70 has a putative RNA recognition motif, the RS-like region, and two coiled-coil domains, suggesting a role in RNA processing. Accordingly, using CD44, Tra2β1 and Fas constructs as splicing reporter minigenes, we found that NSrp70 modulated alternative splice site selection in vivo. The C-terminal 10 amino acids (531-540), including (536)RD(537), were identified as a novel nuclear localization signal, and the region spanning 290-471 amino acids was critical for speckle localization and binding to SC35 and ASF/SF2. The N-terminal region (107-161) was essential for the pre-mRNA splicing activity. Finally, we found that knockout of NSrp70 gene in mice led to a lack of progeny, including fetal embryos. Collectively, we demonstrate that NSrp70 is a novel splicing regulator and essentially required early stage of embryonic development.     

SRSF2 Is Essential for Hematopoiesis,and Its Myelodysplastic Syndrome-Related Mutations Dysregulate Alternative Pre-mRNA Splicing

Myelodysplastic syndromes (MDS) are a group of neoplasms characterized by ineffective myeloid hematopoiesis and various risks for leukemia. SRSF2, a member of the serine/arginine-rich (SR) family of splicing factors, is one of the mutation targets associated with poor survival in patients suffering from myelodysplastic syndromes. Here we report the biological function of SRSF2 in hematopoiesis by using conditional knockout mouse models. Ablation of SRSF2 in the hematopoietic lineage caused embryonic lethality, and Srsf2-deficient fetal liver cells showed significantly enhanced apoptosis and decreased levels of hematopoietic stem/progenitor cells. Induced ablation of SRSF2 in adult Mx1-Cre Srsf2^flox/flox mice upon poly(I):poly(C) injection demonstrated a significant decrease in lineage⁻ Sca⁺ c-Kit⁺ cells in bone marrow. To reveal the functional impact of myelodysplastic syndromes-associated mutations in SRSF2, we analyzed splicing responses on the MSD-L cell line and found that the missense mutation of proline 95 to histidine (P95H) and a P95-to-R102 in-frame 8-amino-acid deletion caused significant changes in alternative splicing. The affected genes were enriched in cancer development and apoptosis. These findings suggest that intact SRSF2 is essential for the functional integrity of the hematopoietic system and that its mutations likely contribute to development of myelodysplastic syndromes.     

A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

The human MAPT locus generates circular RNAs

The microtubule-associated protein Tau, generated by the MAPT gene is involved in dozens of neurodegenerative conditions (“tauopathies”), including Alzheimer's disease (AD) and frontotemporal lobar degeneration/frontotemporal dementia (FTLD/FTD). The pre-mRNA of MAPT is well studied and its aberrant pre-mRNA splicing is associated with frontotemporal dementia. Using a PCR screen of RNA from human brain tissues, we found that the MAPT locus generates circular RNAs through a backsplicing mechanism from exon 12 to either exon 10 or 7. MAPT circular RNAs are localized in the cytosol and contain open reading frames encoding Tau protein fragments. The MAPT exon 10 is alternatively spliced and proteins involved in its regulation, such as CLK2, SRSF7/9G8, PP1 (protein phosphatase 1) and NIPP1 (nuclear inhibitor of PP1) reduce the abundance of the circular MAPT exon 12?→?10 backsplice RNA after being transfected into cultured HEK293 cells. In summary, we report the identification of new bona fide human brain RNAs produced from the MAPT locus. These may be a component of normal human brain Tau regulation and, since the circular RNAs could generate high molecular weight proteins with multiple microtubule binding sites, they could contribute to taupathies.     

Role of the Modular Domains of SR Proteins in Subnuclear Localization and Alternative Splicing Specificity

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5′ splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.     

SANS (USH1G) regulates pre-mRNA splicing by mediating the intra-nuclear transfer of tri-snRNP complexes

Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome—the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.     

Identification of an RNA binding specificity for the potential splicing factor TLS

The TLS/FUS gene is involved in a recurrent chromosomal translocation in human myxoid liposarcomas. We previously reported that TLS is a potential splicing regulator able to modulate the 5'-splice site selection in an E1A pre-mRNA. Using an in vitro selection procedure, we investigated whether TLS exhibits a specificity with regard to RNA recognition. The RNAs selected by TLS share a common GGUG motif. Mutation of a G or U residue within this motif abolishes the interaction of TLS with the selected RNAs. We showed that TLS can bind GGUG-containing RNAs with a 250 nm affinity. By UV cross-linking/competition and immunoprecipitation experiments, we demonstrated that TLS recognizes a GGUG-containing RNA in nuclear extracts. Each one of the RNA binding domains (the three RGG boxes and the RNA recognition motif) contributes to the specificity of the TLS.RNA interaction, whereas only RRM and RGG2-3 participate to the E1A alternative splicing in vivo. The specificity of the TLS.RNA interaction was also observed using as natural pre-mRNA, the G-rich IVSB7 intron of the beta-tropomyosin pre-mRNA. Moreover, we determined that RNA binding specificities of TLS and high nuclear ribonucleoprotein A1 were different. Hence, our results help define the role of the specific interaction of TLS with RNA during the splicing process of a pre-mRNA.