Splicing Enhancers at Intron-Exon Borders Participate in Acceptor Splice Sites Recognition.

Acceptor splice site recognition (3' splice site: 3'ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3'ss, of which U2AF35 has a dual function: (i) It binds to the intron-exon border of some 3'ss and (ii) mediates enhancer-binding splicing activators' interactions with the spliceosome. Alternative mechanisms for 3'ss recognition have been suggested, yet they are still not thoroughly understood. Here, we analyzed 3'ss recognition where the intron-exon border is bound by a ubiquitous splicing regulator SRSF1. Using the minigene analysis of two model exons and their mutants, BRCA2 exon 12 and VARS2 exon 17, we showed that the exon inclusion correlated much better with the predicted SRSF1 affinity than 3'ss quality, which were assessed using the Catalog of Inferred Sequence Binding Preferences of RNA binding proteins (CISBP-RNA) database and maximum entropy algorithm (MaxEnt) predictor and the U2AF35 consensus matrix, respectively. RNA affinity purification proved SRSF1 binding to the model 3'ss. On the other hand, knockdown experiments revealed that U2AF35 also plays a role in these exons' inclusion. Most probably, both factors stochastically bind the 3'ss, supporting exon recognition, more apparently in VARS2 exon 17. Identifying splicing activators as 3'ss recognition factors is crucial for both a basic understanding of splicing regulation and human genetic diagnostics when assessing variants' effects on splicing.

Quantitative Activity Profile and Context Dependence of All Human 5' Splice Sites.

Pre-mRNA splicing is an essential step in the expression of most human genes. Mutations at the 5' splice site (5'ss) frequently cause defective splicing and disease due to interference with the initial recognition of the exon-intron boundary by U1 small nuclear ribonucleoprotein (snRNP), a component of the spliceosome. Here, we use a massively parallel splicing assay (MPSA) in human cells to quantify the activity of all 32,768 unique 5'ss sequences (NNN/GYNNNN) in three different gene contexts. Our results reveal that although splicing efficiency is mostly governed by the 5'ss sequence, there are substantial differences in this efficiency across gene contexts. Among other uses, these MPSA measurements facilitate the prediction of 5'ss sequence variants that are likely to cause aberrant splicing. This approach provides a framework to assess potential pathogenic variants in the human genome and streamline the development of splicing-corrective therapies.

Splicing factors of SR and hnRNP families as regulators of apoptosis in cancer.

SR and hnRNP proteins were initially discovered as regulators of alternative splicing: the process of controlled removal of introns and selective joining of exons through which multiple transcripts and, subsequently, proteins can be expressed from a single gene. Alternative splicing affects genes involved in all crucial cellular processes, including apoptosis. During cancerogenesis impaired apoptotic control facilitates survival of cells bearing molecular aberrations, contributing to their unrestricted proliferation and chemoresistance. Apparently, SR and hnRNP proteins regulate all levels of expression of apoptotic genes, including transcription initiation and elongation, alternative splicing, mRNA stability, translation, and protein degradation. The frequently disturbed expressions of SR/hnRNP proteins in cancers lead to impaired functioning of target apoptotic genes, including regulators of the extrinsic (Fas, caspase-8, caspase-2, c-FLIP) and the intrinsic pathway (Apaf-1, caspase-9, ICAD), genes encoding Bcl-2 proteins, IAPs, and p53 tumor suppressor. Prototypical members of SR/hnRNP families, SRSF1 and hnRNP A1, promote synthesis of anti-apoptotic splice variants of Bcl-x and Mcl-1, which results in attenuation of programmed cell death in breast cancer and chronic myeloid leukemia. SR/hnRNP proteins significantly affect responses to chemotherapy, acting as mediators or modulators of drug-induced apoptosis. Aberrant expression of SRSF1 and hnRNP K can interfere with tumor responses to chemotherapy in pancreatic and liver cancers. Currently, a number of splicing factor inhibitors is being tested in pre-clinical and clinical trials. In this review we discuss recent findings on the role of SR and hnRNP proteins in apoptotic control in cancer cells as well as their significance in anticancer treatments.

Suppression of 5' splice-sites through multiple exonic motifs by hnRNP L.

Selection of 5' splice-sites (5'SS) in alternative splicing plays an important role in gene regulation. Although regulatory mechanisms of heterogeneous nuclear ribonucleoprotein L (hnRNP L), a well-known splicing regulatory protein, have been studied in a substantial level, its role in 5'SS selection is not thoroughly defined. By using a KLF6 pre-mRNA alternative splicing model, we demonstrate in this report that hnRNP L inhibits proximal 5'SS but promotes two consecutive distal 5'SS splicing, antagonizing SRSF1 roles in KLF6 pre-mRNA splicing. In addition, three consecutive CA-rich sequences in a CA cassette immediately upstream of the proximal 5'SS are all required for hnRNP L functions. Importantly, the CA-cassette locations on the proximal exon do not affect hnRNP L roles. We further show that the proximal 5'SS but not the two distal 5'SSs are essential for hnRNP L activities. Notably, in a Bcl-x pre-mRNA model that contains two alternative 5'SS but includes CA-rich elements at distal exon, we demonstrate that hnRNP L also suppresses nearby 5'SS activation. Taken together, we conclude that hnRNP L suppresses 5'SS selection through multiple exonic motifs.

How slow RNA polymerase II elongation favors alternative exon skipping.

Splicing is functionally coupled to transcription, linking the rate of RNA polymerase II (Pol II) elongation and the ability of splicing factors to recognize splice sites (ss) of various strengths. In most cases, slow Pol II elongation allows weak splice sites to be recognized, leading to higher inclusion of alternative exons. Using CFTR alternative exon 9 (E9) as a model, we show here that slowing down elongation can also cause exon skipping by promoting the recruitment of the negative factor ETR-3 onto the UG-repeat at E9 3' splice site, which displaces the constitutive splicing factor U2AF65 from the overlapping polypyrimidine tract. Weakening of E9 5' ss increases ETR-3 binding at the 3' ss and subsequent E9 skipping, whereas strengthening of the 5' ss usage has the opposite effect. This indicates that a delay in the cotranscriptional emergence of the 5' ss promotes ETR-3 recruitment and subsequent inhibition of E9 inclusion.

The DEAH box ATPases Prp16 and Prp43 cooperate to proofread 5' splice site cleavage during pre-mRNA splicing.

To investigate the mechanisms underlying accurate pre-mRNA splicing, we developed an in vitro assay sensitive to proofreading of 5' splice site cleavage. We inactivated spliceosomes by disrupting a metal-ligand interaction at the catalytic center and discovered that, when the DEAH box ATPase Prp16 was disabled, these spliceosomes catalyzed 5' splice site cleavage but at a reduced rate. Although Prp16 does not promote splicing of a genuine substrate until after 5' splice site cleavage, we found that Prp16 can associate with spliceosomes before 5' splice site cleavage, consistent with a role for Prp16 in proofreading 5' splice site cleavage. We established that Prp16-mediated rejection is reversible, necessitating a downstream discard pathway that we found requires the DEAH box ATPase Prp43, a spliceosome disassembly factor. These data indicate that spliceosomes distinguish slow substrates and that the mechanisms for establishing the fidelity of 5' splice site cleavage and exon ligation share a common ATP-dependent framework.

Characterization of an alternative splice variant of LKB1.

LKB1 is an upstream activating kinase for the AMP-activated protein kinase (AMPK) and at least 12 other AMPK-related kinases. LKB1 therefore acts as a master kinase regulating the activity of a wide range of downstream kinases, which themselves have diverse physiological roles. Here we identify a second form of LKB1 generated by alternative splicing of the LKB1 gene. The two LKB1 proteins have different C-terminal sequences generating a 50-kDa form (termed LKB1L) and a 48-kDa form (LKB1S). LKB1L is widely expressed in mouse tissues, whereas LKB1S has a restricted tissue distribution with predominant expression in the testis. LKB1S, like LKB1L, forms a complex with MO25 and STRAD, and phosphorylates and activates AMPK both in vitro and in intact cells. A phosphorylation site (serine 431 in mouse) and a farnesylation site (cysteine 433 in mouse) within LKB1L are not conserved in LKB1S raising the possibility that these sites might be involved in differential regulation and/or localization of the two forms of LKB1. However, we show that phosphorylation of serine 431 has no effect on LKB1L activity and that both LKB1L and LKB1S have similar patterns of subcellular localization. These results indicate that the physiological significance of the different forms of LKB1 is not related directly to differences in the C-terminal sequences but may be due to their differential patterns of tissue distribution.

Solution conformation and thermodynamic characteristics of RNA binding by the splicing factor U2AF65.

The U2 auxiliary factor large subunit (U2AF65) is an essential pre-mRNA splicing factor for the initial stages of spliceosome assembly. Tandem RNA recognition motifs (RRM)s of U2AF65 recognize polypyrimidine tract signals adjacent to 3' splice sites. Despite the central importance of U2AF65 for splice site recognition, the relative arrangement of the U2AF65 RRMs and the energetic forces driving polypyrimidine tract recognition remain unknown. Here, the solution conformation of the U2AF65 RNA binding domain determined using small angle x-ray scattering reveals a bilobal shape without apparent interdomain contacts. The proximity of the N and C termini within the inter-RRM configuration is sufficient to explain the action of U2AF65 on spliceosome components located both 5' and 3' to its binding site. Isothermal titration calorimetry further demonstrates that an unusually large enthalpy-entropy compensation underlies U2AF65 recognition of an optimal polyuridine tract. Qualitative similarities were observed between the pairwise distance distribution functions of the U2AF65 RNA binding domain and those either previously observed for N-terminal RRMs of Py tract-binding protein that lack interdomain contacts or calculated from the high resolution coordinates of a U2AF65 deletion variant bound to RNA. To further test this model, the shapes and RNA interactions of the wild-type U2AF65 RNA binding domain were compared with those of U2AF65 variants containing either Py tract-binding protein linker sequences or a deletion within the inter-RRM linker. Results of these studies suggest inter-RRM conformational plasticity as a possible means for U2AF65 to universally identify diverse pre-mRNA splice sites.

Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors.

Vascular endothelial growth factor A (VEGFA; hereafter referred to as VEGF) is a key regulator of physiological and pathological angiogenesis. Two families of VEGF isoforms are generated by alternate splice-site selection in the terminal exon. Proximal splice-site selection (PSS) in exon 8 results in pro-angiogenic VEGFxxx isoforms (xxx is the number of amino acids), whereas distal splice-site selection (DSS) results in anti-angiogenic VEGFxxxb isoforms. To investigate control of PSS and DSS, we investigated the regulation of isoform expression by extracellular growth factor administration and intracellular splicing factors. In primary epithelial cells VEGFxxxb formed the majority of VEGF isoforms (74%). IGF1, and TNFalpha treatment favoured PSS (increasing VEGFxxx) whereas TGFbeta1 favoured DSS, increasing VEGFxxxb levels. TGFbeta1 induced DSS selection was prevented by inhibition of p38 MAPK and the Clk/sty (CDC-like kinase, CLK1) splicing factor kinase family, but not ERK1/2. Clk phosphorylates SR protein splicing factors ASF/SF2, SRp40 and SRp55. To determine whether SR splicing factors alter VEGF splicing, they were overexpressed in epithelial cells, and VEGF isoform production assessed. ASF/SF2, and SRp40 both favoured PSS, whereas SRp55 upregulated VEGFxxxb (DSS) isoforms relative to VEGFxxx. SRp55 knockdown reduced expression of VEGF165b. Moreover, SRp55 bound to a 35 nucleotide region of the 3'UTR immediately downstream of the stop codon in exon 8b. These results identify regulation of splicing by growth and splice factors as a key event in determining the relative pro-versus anti-angiogenic expression of VEGF isoforms, and suggest that p38 MAPK-Clk/sty kinases are responsible for the TGFbeta1-induced DSS selection, and identify SRp55 as a key regulatory splice factor.

Hu antigen R (HuR) functions as an alternative pre-mRNA splicing regulator of Fas apoptosis-promoting receptor on exon definition.

Exclusion of exon 6 by alternative RNA splicing of the primary transcript of the apoptosis receptor Fas produces a soluble isoform that prevents programmed cell death. I report that antiapoptotic regulator Hu antigen R (HuR, ELAVL1), a member of the embryonic lethal, abnormal vision, Drosophila-like (ELAVL) family, promotes Fas exon 6 skipping by binding to an exonic splicing silencer. HuR inhibits the association of U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 kDa (U2AF65) with the upstream 3' splice site, without decreasing recognition of the downstream 5' splice site by U1 snRNP but by antagonizing the role of TIA-1 (T-cell intracellular antigen 1)/TIAR (TIA-1 related protein) on exon definition. Remarkably, U1 snRNP-mediated recognition of the 5' splice site is partially required for efficient U2AF65 inhibition. Further, the silencing capacity of HuR as splicing regulator resides in the RRM1 and hinge-RRM3 domains. Taken together, these results support a functional link between HuR as repressor of alternative Fas splicing and the molecular mechanisms modulating programmed cell death.

Pre-mRNA splicing: a complex picture in higher definition.

Intron excision from pre-mRNAs of higher eukaryotes requires a transition from splice-site recognition across short exons to organization of the spliceosome across long introns. Recently, insight into this transition has been provided and, in addition, it has been shown that an alternative splicing factor, the polypyrimidine-tract-binding protein, can exert its control on splice-site choice by blocking this key step in the assembly of the splicing machinery.

Alternative splicing and disease.

Splicing is a molecular mechanism, by which introns are removed from an mRNA precursor and exons are ligated to form a mature mRNA. Mutations that cause defects in the splicing mechanism are known to be responsible for many diseases, including cystic fibrosis and familial dysautonomia. If mutations that cause defects in splicing are responsible for such severe deleterious phenotypic differences, it is possible that mutations in splicing are also responsible for mildly deleterious phenotypic differences. Although deleterious mutations are rapidly eliminated from the population by purifying selection, the selection against mild deleterious effects is not as strong. Since mildly deleterious mutations have a chance of surviving natural selection, we might be mistakenly referring to these mutations as neutral variation between individuals. Splicing has also been shown to be seriously affected in cancer. Examination of cancerous tissues revealed alterations in expression levels of genes involved in mRNA processing and also a slight reduction in the level of exon skipping--the most common form of alternative splicing in humans. This implies that defects in genes involved in the regulation of splicing in cancerous tissues affect the delicate regulation of the inclusion level of alternatively skipped exons, shifting their mode of splicing back to constitutive. It may be that splicing silencers play a more prominent role in alternative splicing regulation than previously anticipated.

Retroviral splicing suppressor sequesters a 3' splice site in a 50S aberrant splicing complex.

Retroviral replication requires both spliced and unspliced mRNAs. Splicing suppression of avian retroviral RNA depends in part upon a cis-acting element within the gag gene called the negative regulator of splicing (NRS). The NRS, linked to a downstream intron and exon (NRS-Ad3'), was not capable of splicing in vitro. However, a double-point mutation in the NRS pseudo-5' splice site sequence converted it into a functional 5' splice site. The wild-type (WT) NRS-Ad3' transcript assembled an approximately 50S spliceosome-like complex in vitro; its sedimentation rate was similar to that of a functional spliceosome formed on the mutant NRS-Ad3' RNA. The five major spliceosomal snRNPs were observed in both complexes by affinity selection. In addition, U11 snRNP was present only in the WT NRS-Ad3' complex. Addition of heparin to these complexes destabilized the WT NRS-Ad3' complex; it was incapable of forming a B complex on a native gel. Furthermore, the U5 snRNP protein, hPrp8, did not cross-link to the NRS pseudo-5' splice site, suggesting that the tri-snRNP complex was not properly associated with it. We propose that this aberrant, stalled spliceosome, containing U1, U2, and U11 snRNPs and a loosely associated tri-snRNP, sequesters the 3' splice site and prevents its interaction with the authentic 5' splice site upstream of the NRS.

Substrate-dependent differences in U2AF requirement for splicing in adenovirus-infected cell extracts.

U2AF has been characterized as an essential splicing factor required for efficient recruitment of U2 small nuclear ribonucleoprotein to the 3'-splice site in a pre-mRNA. The U2AF65 subunit binds to the pyrimidine tract of the pre-mRNA, whereas the U2AF(35) subunit contacts the 3'-splice site AG. Here we show that U2AF35 appears to be completely dispensable for splicing in nuclear extracts prepared from adenovirus late-infected cells (Ad-NE). As a consequence, the viral IIIa and cellular IgM introns, which both have suboptimal 3'-splice sites and require U2AF35 for splicing in nuclear extracts from uninfected cells, are transformed to U2AF35-independent introns in Ad-NE. Furthermore, we present evidence that two parallel pathways of 3'-splice site recognition exist in Ad-NE. We show that the viral 52,55K intron, which has an extended pyrimidine tract, requires U2AF for activity in Ad-NE. In contrast, the IgM intron, which has a weak 3'-splice site sequence context, undergoes the first catalytic step of splicing in U2AF-depleted Ad-NE, suggesting that spliceosome assembly occurs through a novel U2AF-independent pathway in Ad-NE.

New strategies for exploring RNA's 2'-OH expose the importance of solvent during group II intron catalysis.

The 2'-hydroxyl group contributes inextricably to the functional behavior of many RNA molecules, fulfilling numerous essential chemical roles. To assess how hydroxyl groups impart functional behavior to RNA, we developed a series of experimental strategies using an array of nucleoside analogs. These strategies provide the means to investigate whether a hydroxyl group influences function directly (via hydrogen bonding or metal ion coordination), indirectly (via space-filling capacity, inductive effects, and sugar conformation), or through interactions with solvent. The nucleoside analogs span a broad range of chemical diversity, such that quantitative structure activity relationships (QSAR) now become possible in the exploration of RNA biology. We employed these strategies to investigate the spliced exons reopening (SER) reaction of the group II intron. Our results suggest that the cleavage site 2'-hydroxyl may mediate an interaction with a water molecule.

The second RNA-binding domain of the human splicing factor ASF/SF2 is the critical domain controlling adenovirus E1A alternative 5'-splice site selection.

The human splicing factor ASF/SF2 (alternative splicing factor/splicing factor 2) is modular in structure with two RNA-binding domains (RBD1 and RBD2) and a C-terminal domain rich in arginine-serine dipeptide repeats. ASF/SF2 is an essential splicing factor that also functions as an important regulator of alternative splicing. In adenovirus E1A (early region 1A) alternative pre-mRNA splicing, ASF/SF2 functions as a strong inducer of proximal 5'-splice-site selection, both in vitro and in vivo. In the present study, we tested the functional role of individual domains of ASF/SF2 in alternative splicing in vitro. We show that ASF/SF2-RBD2 is the critical domain controlling E1A alternative splicing. In fact, RBD2 alone is sufficient to mimic the activity of the full-length ASF/SF2 protein as an inducer of proximal 5'-splice-site selection in vitro. The RBD2 domain induces a switch to E1A-proximal 5'-splice-site usage by repressing distal 12 S splicing and simultaneously stimulates proximal 13 S splicing. In contrast, the ASF/SF2-RBD1 domain has a more general splicing enhancer phenotype and appears to stimulate preferentially cap-proximal 5'-splice-site selection. Furthermore, the SWQDLKD motif, which is conserved in all SR proteins (serine/arginine-rich proteins) containing two RBDs, and the ribonucleoprotein-1-type RNA recognition motif were both found to be necessary for the alternative splice-site-switching activity of ASF/SF2. The RNP-1 motif was necessary for efficient RNA binding, whereas the SWQDLKD motif most probably contributes by functioning as a surface-mediating critical protein-protein contact during spliceosome assembly.

Changes in the number of HSF1 positive granules in the nucleus reflects heat shock semiquantitatively.

PURPOSE: The purpose of this study is to examine the changes in the number of HSF1 granules in the nucleus caused by different degree of heat stress. MATERIALS AND METHODS: A human esophageal cancer cell line, TE-2, was used. HSF1 granules were examined in an immunofluorescence study, and the changes in the average number of HSF1 granules after heat alone or heat in combination with KNK437, Hsp inhibitor, were evaluated. A band shift of HSF1 was also determined by western blot. RESULTS: HSF1 granules appeared soon after the start of heating at 43 degrees C and reached a peak at 60 min and gradually disappeared after discontinuation of heat. In the fractionated heat treatment, preheating (43 degrees C, 30 min) suppressed the increase in the number of the granules during the second heating, but suppression of Hsp72 by KNK437 resulted in increase in the number of granules. Continued heating at 43 degrees C with or without KNK437 maintained the number of the granules at the peak level during heat treatment. The band shift of HSF1 examined by western blot correlated with the changes in the number of granules. The number of granules also reflected the degree of stress according to different temperature. CONCLUSION: The number of HSF1 granules in the nucleus well reflected heat stress, and was almost consistent with phosphorylation of HSF1. The number of HSF1 granules would be a useful tool for evaluating different degrees of heat stress semiquantitatively.

Alternative splicing of the human estrogen receptor alpha primary transcript: mechanisms of exon skipping.

The 1785 nucleotides of the coding region of the estrogen receptor alpha (ER-alpha) are dispersed over a region of more than 300,000 nucleotides in the primary transcript. Splicing of this precursor RNA frequently leads to variants lacking one or more exons that have been associated to breast cancer progression. The most frequent splice variant lacks exon 4 and is expressed in the human mammary carcinoma cell line MCF-7 at a level similar to that of the full-length messenger. The in silico analysis of ER-alpha splice sites by Hamming clustering, a self learning method trained on more than 28,000 experimentally proved splice sites, reveals high relevance for the 5' and 3' splice sites of exon 4. The splicing analysis of transfected mini-gene constructs containing drastically shortened introns excludes that weak splice sites, intron or exon lengths or splice enhancers are responsible for exon skipping. Exon 6 is never skipped in MCF-7 cells but is spliced out from mini-gene derived primary transcripts if inserted between exons 3 and 5 instead of exon 4. As a consequence, it appears that a particular splice site affinity of exon 3 donor (5' splice site) and exon 5 acceptor sites (3' splice site) is responsible for skipping of the exon in between.

Binding of a SART3 tumor-rejection antigen to a pre-mRNA splicing factor RNPS1: a possible regulation of splicing by a complex formation.

We recently reported the identification of a human SART3 gene that encodes a tumor-rejection antigen recognized by cytotoxic T lymphocytes (CTLs). The squamous-cell carcinoma antigen recognized by T cells-3 (SART3) is an RNA-binding protein expressed in the nucleus of the majority of proliferating cells, including normal cells and malignant cells, but not in normal tissues except for the testes and fetal liver. To determine its biologic function, we employed a 2-hybrid screening in yeast for proteins interacting with SART3, and this method yielded a pre-mRNA splicing factor (RNA-binding protein prevalent during the S phase or RNA-binding protein with a serine-rich domain [RNPS1]) that activated both constitutive and alternative splicing of pre-mRNA in vitro. Interaction of SART3 with RNPS1 through the physical association of N-terminal domains of RNPS1 was confirmed by both in vitro pull-down assay and immunoprecipitation assay. Cotransfection of the 2 genes changed the distribution pattern of SART3 from diffuse nucleoplasmic spreading to nuclear speckled regions in which the RNPS1 was colocalized, suggesting a complex formation of the 2 proteins. In cooperation with RNPS1, SART3 stimulated the proximal alternative 3' splicing of a calcitonin-dihydrofolate reductase chimeric minigene pre-mRNA. These results suggest that SART3 is involved in the regulation of mRNA splicing probably via its complex formation with RNPS1.