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1.  An Exonic Splicing Silencer Downstream of the 3′ Splice Site A2 Is Required for Efficient Human Immunodeficiency Virus Type 1 Replication 
Journal of Virology  2005;79(16):10478-10486.
Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic mRNA produces more than 40 unique viral mRNA species, of which more than half remain incompletely spliced within an HIV-1-infected cell. Regulation of splicing at HIV-1 3′ splice sites (3′ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation of splicing occurs through binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3′ss A2, which produces vpr mRNA and promotes inclusion of HIV-1 exon 3, is repressed by the hnRNP A/B-dependent exonic splicing silencer ESSV. Here we show that ESSV activity downstream of 3′ss A2 is localized to a 16-nucleotide element within HIV-1 exon 3. HIV-1 replication was reduced by 95% when ESSV was inactivated by mutagenesis. Reduced replication was concomitant with increased inclusion of exon 3 within spliced viral mRNA and decreased accumulation of unspliced viral mRNA, resulting in decreased cell-associated p55 Gag. Prolonged culture of ESSV mutant viruses resulted in two independent second-site reversions disrupting the splice sites that define exon 3, 3′ss A2 and 5′ splice site D3. Either of these changes restored both HIV-1 replication and regulated viral splicing. Therefore, inhibition of HIV-1 3′ss A2 splicing is necessary for HIV-1 replication.
PMCID: PMC1182660  PMID: 16051840
2.  RNA Splicing at Human Immunodeficiency Virus Type 1 3′ Splice Site A2 Is Regulated by Binding of hnRNP A/B Proteins to an Exonic Splicing Silencer Element 
Journal of Virology  2001;75(18):8487-8497.
The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3′ splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1B, A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore splicing repression. The results suggest that coordinate repression of HIV-1 RNA splicing is mediated by members of the hnRNP A/B protein family.
PMCID: PMC115094  PMID: 11507194
3.  A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication 
Retrovirology  2006;3:10.
Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1) primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss), which are positively regulated by exonic splicing enhancers (ESE) and negatively regulated by exonic splicing silencers (ESS) and intronic splicing silencers (ISS). We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production.
Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3.
These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non-coding exon inclusion in HIV-1 replication.
PMCID: PMC1403798  PMID: 16457729
4.  Cooperative-Binding and Splicing-Repressive Properties of hnRNP A1▿ † 
Molecular and Cellular Biology  2009;29(20):5620-5631.
hnRNP A1 binds to RNA in a cooperative manner. Initial hnRNP A1 binding to an exonic splicing silencer at the 3′ end of human immunodeficiency virus type 1 (HIV-1) tat exon 3, which is a high-affinity site, is followed by cooperative spreading in a 3′-to-5′ direction. As hnRNP A1 propagates toward the 5′ end of the exon, it antagonizes binding of a serine/arginine-rich (SR) protein to an exonic splicing enhancer, thereby inhibiting splicing at that exon's alternative 3′ splice site. tat exon 3 and the preceding intron of HIV-1 pre-mRNA can fold into an elaborate RNA secondary structure in solution, which could potentially influence hnRNP A1 binding. We report here that hnRNP A1 binding and splicing repression can occur on an unstructured RNA. Moreover, hnRNP A1 can effectively unwind an RNA hairpin upon binding, displacing a bound protein. We further show that hnRNP A1 can also spread in a 5′-to-3′ direction, although when initial binding takes place in the middle of an RNA, spreading preferentially proceeds in a 3′-to-5′ direction. Finally, when two distant high-affinity sites are present on the same RNA, they facilitate cooperative spreading of hnRNP A1 between the two sites.
PMCID: PMC2756886  PMID: 19667073
5.  Identification of an hnRNP A1-Dependent Splicing Silencer in the Human Papillomavirus Type 16 L1 Coding Region That Prevents Premature Expression of the Late L1 Gene 
Journal of Virology  2004;78(20):10888-10905.
We have previously identified cis-acting RNA sequences in the human papillomavirus type 16 (HPV-16) L1 coding region which inhibit expression of L1 from eukaryotic expression plasmids. Here we have determined the function of one of these RNA elements, and we provide evidence that this RNA element is a splicing silencer which suppresses the use of the 3′ splice site located immediately upstream of the L1 AUG. We also show that this splice site is inefficiently utilized as a result of a suboptimal polypyrimidine tract. Introduction of point mutations in the L1 coding region that altered the RNA sequence without affecting the L1 protein sequence resulted in the inactivation of the splicing silencer and induced splicing to the L1 3′ splice site. These mutations also prevented the interaction of the RNA silencer with a 35-kDa cellular protein identified here as hnRNP A1. The splicing silencer in L1 inhibits splicing in vitro, and splicing can be restored by the addition of RNAs containing an hnRNP A1 binding site to the reaction, demonstrating that hnRNP A1 inhibits splicing of the late HPV-16 mRNAs through the splicing silencer sequence. While we show that one role of the splicing silencer is to determine the ratio between partially spliced L2/L1 mRNAs and spliced L1 mRNAs, we also demonstrate that it inhibits splicing from the major 5′ splice site in the early region to the L1 3′ splice site, thereby playing an essential role in preventing late gene expression at an early stage of the viral life cycle. We speculate that the activity of the splicing silencer and possibly the concentration of hnRNP A1 in the HPV-16-infected cell determines the ability of the virus to establish a persistent infection which remains undetected by the host immune surveillance.
PMCID: PMC521837  PMID: 15452209
6.  hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells 
Molecular and Cellular Biology  1999;19(1):69-77.
The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5′ splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chromatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide sequence analysis of purified p58 protein identified it as hnRNP H. Immunoprecipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel mobility shift analysis of the enhancer complex in the presence of hnRNP H-specific antibodies, confirmed that hnRNP H is a protein component of the splicing enhancer complex. Immunoprecipitation of splicing intermediates from in vitro splicing reactions with anti-hnRNP H antibody indicated that hnRNP H remains bound to the src pre-mRNA after the assembly of spliceosome. Partial immunodepletion of hnRNP H from the nuclear extract partially inactivated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP H, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H can interact with the related protein hnRNP F, suggesting that hnRNPs H and F may exist as a heterodimer in a single enhancer complex. These two proteins presumably cooperate with each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream.
PMCID: PMC83866  PMID: 9858532
7.  hnRNP A1 Recruited to an Exon In Vivo Can Function as an Exon Splicing Silencer 
Molecular and Cellular Biology  1999;19(1):251-260.
Some exons contain exon splicing silencers. Their activity is frequently balanced by that of splicing enhancers, and this is important to ensure correct relative levels of alternatively spliced mRNAs. Using an immunoprecipitation and UV-cross-linking assay, we show that RNA molecules containing splicing silencers from the human immunodeficiency virus type 1 tat exon 2 or the human fibroblast growth factor receptor 2 K-SAM exon bind to hnRNP A1 in HeLa cell nuclear extracts better than the corresponding RNA molecule without a silencer. Two different point mutations which abolish the K-SAM exon splicing silencer’s activity reduce hnRNP A1 binding twofold. Recruitment of hnRNP A1 in the form of a fusion with bacteriophage MS2 coat protein to a K-SAM exon whose exon splicing silencer has been replaced by a coat binding site efficiently represses splicing of the exon in vivo. Recruitment of only the glycine-rich C-terminal domain of hnRNP A1, which is capable of interactions with other proteins, is sufficient to repress exon splicing. Our results show that hnRNP A1 can function to repress splicing, and they suggest that at least some exon splicing silencers could work by recruiting hnRNP A1.
PMCID: PMC83883  PMID: 9858549
8.  General and Specific Functions of Exonic Splicing Silencers in Splicing Control 
Molecular cell  2006;23(1):61-70.
Correct splice site recognition is critical in pre-mRNA splicing. We find that almost all of a diverse panel of exonic splicing silencer (ESS) elements alter splice site choice when placed between competing sites, consistently inhibiting use of intron-proximal 5′ and 3′ splice sites. Supporting a general role for ESSs in splice site definition, we found that ESSs are both abundant and highly conserved between alternative splice site pairs and that mutation of ESSs located between natural alternative splice site pairs consistently shifted splicing toward the intron-proximal site. Some exonic splicing enhancers (ESEs) promoted use of intron-proximal 5′ splice sites, and tethering of hnRNP A1 and SF2/ASF proteins between competing splice sites mimicked the effects of ESS and ESE elements, respectively. Further, we observed that specific subsets of ESSs had distinct effects on a multifunctional intron retention reporter, and that one of these subsets is likely preferred for regulation of endogenous intron retention events. Together, our findings provide a comprehensive picture of the functions of ESSs in the control of diverse types of splicing decisions.
PMCID: PMC1839040  PMID: 16797197
9.  HnRNP L and hnRNP LL antagonistically modulate PTB-mediated splicing suppression of CHRNA1 pre-mRNA 
Scientific Reports  2013;3:2931.
CHRNA1 gene, encoding the muscle nicotinic acetylcholine receptor alpha subunit, harbors an inframe exon P3A. Inclusion of exon P3A disables assembly of the acetylcholine receptor subunits. A single nucleotide mutation in exon P3A identified in congenital myasthenic syndrome causes exclusive inclusion of exon P3A. The mutation gains a de novo binding affinity for a splicing enhancing RNA-binding protein, hnRNP LL, and displaces binding of a splicing suppressing RNA-binding protein, hnRNP L. The hnRNP L binds to another splicing repressor PTB through the proline-rich region and promotes PTB binding to the polypyrimidine tract upstream of exon P3A, whereas hnRNP LL lacking the proline-rich region cannot bind to PTB. Interaction of hnRNP L with PTB inhibits association of U2AF65 and U1 snRNP with the upstream and downstream of P3A, respectively, which causes a defect in exon P3A definition. HnRNP L and hnRNP LL thus antagonistically modulate PTB-mediated splicing suppression of exon P3A.
PMCID: PMC3796306  PMID: 24121633
10.  A functional conserved intronic G run in HIV-1 intron 3 is critical to counteract APOBEC3G-mediated host restriction 
Retrovirology  2014;11(1):72.
The HIV-1 accessory proteins, Viral Infectivity Factor (Vif) and the pleiotropic Viral Protein R (Vpr) are important for efficient virus replication. While in non-permissive cells an appropriate amount of Vif is critical to counteract APOBEC3G-mediated host restriction, the Vpr-induced G2 arrest sets the stage for highest transcriptional activity of the HIV-1 long terminal repeat.
Both vif and vpr mRNAs harbor their translational start codons within the intron bordering the non-coding leader exons 2 and 3, respectively. Intron retention relies on functional cross-exon interactions between splice sites A1 and D2 (for vif mRNA) and A2 and D3 (for vpr mRNA). More precisely, prior to the catalytic step of splicing, which would lead to inclusion of the non-coding leader exons, binding of U1 snRNP to the 5' splice site (5'ss) facilitates recognition of the 3'ss by U2 snRNP and also supports formation of vif and vpr mRNA.
We identified a G run localized deep in the vpr AUG containing intron 3 (GI3-2), which was critical for balanced splicing of both vif and vpr non-coding leader exons. Inactivation of GI3-2 resulted in excessive exon 3 splicing as well as exon-definition mediated vpr mRNA formation. However, in an apparently mutually exclusive manner this was incompatible with recognition of upstream exon 2 and vif mRNA processing. As a consequence, inactivation of GI3-2 led to accumulation of Vpr protein with a concomitant reduction in Vif protein. We further demonstrate that preventing hnRNP binding to intron 3 by GI3-2 mutation diminished levels of vif mRNA. In APOBEC3G-expressing but not in APOBEC3G-deficient T cell lines, mutation of GI3-2 led to a considerable replication defect. Moreover, in HIV-1 isolates carrying an inactivating mutation in GI3-2, we identified an adjacent G-rich sequence (GI3-1), which was able to substitute for the inactivated GI3-2.
The functionally conserved intronic G run in HIV-1 intron 3 plays a major role in the apparently mutually exclusive exon selection of vif and vpr leader exons and hence in vif and vpr mRNA formation. The competition between these exons determines the ability to evade APOBEC3G-mediated antiviral effects due to optimal vif expression.
Electronic supplementary material
The online version of this article (doi:10.1186/s12977-014-0072-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4163160  PMID: 25169827
HIV-1 infection; Host restriction; Cytidine deaminase; APOBEC3G; Viral infectivity factor (Vif); Viral protein R (Vpr); Alternative pre-mRNA splicing; G run, hnRNP F/H; Locked nucleic acids (LNAs)
11.  Negative and Positive mRNA Splicing Elements Act Competitively To Regulate Human Immunodeficiency Virus Type 1 Vif Gene Expression▿  
Journal of Virology  2008;82(8):3921-3931.
Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3′ splice sites (3′ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3′ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5′ splice site (5′ss) downstream of exon 2 (5′ss D2). Here we show that the mutations within 5′ss D2 that are predicted to lower or increase the affinity of the 5′ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5′ss D2 was not necessary for the effect of 5′ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5′ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5′-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5′ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication.
PMCID: PMC2293006  PMID: 18272582
12.  Efficient polyadenylation of Rous sarcoma virus RNA requires the negative regulator of splicing element 
Nucleic Acids Research  2002;30(3):810-817.
Rous sarcoma virus pre-mRNA contains an element known as the negative regulator of splicing (NRS) that acts to inhibit viral RNA splicing. The NRS binds serine/arginine-rich (SR) proteins, hnRNP H and the U1/U11 snRNPs, and appears to inhibit splicing by acting as a decoy 5′ splice site. Deletions within the gag gene that encompass the NRS also lead to increased read-through past the viral polyadenylation site, suggesting a role for the NRS in promoting polyadenylation. Using NRS-specific deletions and mutations, we show here that a polyadenylation stimulatory activity maps directly to the NRS and is most likely dependent upon SR proteins and U1 and/or U11 snRNP. hnRNP H does not appear to mediate splicing control or stimulate RSV polyadenylation, since viral RNAs containing hnRNP H-specific mutations were spliced and polyadenylated normally. However, the ability of hnRNP H mutations to suppress the read-through caused by an SR protein mutation suggests the potential for hnRNP H to antagonize polyadenylation. Interestingly, disruption of splicing control closely correlated with increased read-through, indicating that a functional NRS is necessary for efficient RSV polyadenylation rather than binding of an individual factor. We propose a model in which the NRS serves to enhance polyadenylation of RSV unspliced RNA in a process analogous to the stimulation of cellular pre-mRNA polyadenylation by splicing complexes.
PMCID: PMC100303  PMID: 11809895
13.  Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors. 
Molecular and Cellular Biology  1995;15(8):4606-4615.
Human immunodeficiency virus type 1 (HIV-1) pre-mRNA splicing is regulated in order to maintain pools of unspliced and partially spliced viral RNAs as well as the appropriate levels of multiply spliced mRNAs during virus infection. We have previously described an element in tat exon 2 that negatively regulates splicing at the upstream tat 3' splice site 3 (B. A. Amendt, D. Hesslein, L.-J. Chang, and C. M. Stoltzfus, Mol. Cell. Biol. 14:3960-3970, 1994). In this study, we further defined the element to a 20-nucleotide (nt) region which spans the C-terminal vpr and N-terminal tat coding sequences. By analogy with exon splicing enhancer (ESE) elements, we have termed this element an exon splicing silencer (ESS). We show evidence for another negative cis-acting region within tat-rev exon 3 of HIV-1 RNA that has sequence motifs in common with a 20-nt ESS element in tat exon 2. This sequence is juxtaposed to a purine-rich ESE element to form a bipartite element regulating splicing at the upstream tat-rev 3' splice site. Inhibition of the splicing of substrates containing the ESS element in tat exon 2 occurs at an early stage of spliceosome assembly. The inhibition of splicing mediated by the ESS can be specifically abrogated by the addition of competitor RNA. Our results suggest that HIV-1 RNA splicing is regulated by cellular factors that bind to positive and negative cis elements in tat exon 2 and tat-rev exon 3.
PMCID: PMC230701  PMID: 7623852
14.  Polypyrimidine tract-binding protein and heterogeneous nuclear ribonucleoprotein A1 bind to human T-cell leukemia virus type 2 RNA regulatory elements. 
Journal of Virology  1995;69(11):6852-6858.
Efficient expression of human T-cell leukemia virus (HTLV) and human immunodeficiency virus structural proteins requires Rx and Rev proteins, respectively. Decreased expression of Gag and Env appears to be due, in part, to intragenic RNA sequences, termed cis-acting repressive sequences (CRS), and may be mediated by binding of specific cellular factors. We demonstrated previously that two cellular proteins, p60CRS and p40CRS, interact with HTLV type 2.5' long terminal repeat CRS RNA and that the interaction of both proteins with CRS RNA correlates with function (A. C. Black, C. T. Ruland, J. Luo, A. Bakker, J. K. Fraser, and J. D. Rosenblatt, Virology 200:29-41, 1994). By radioimmunoprecipitation of HeLa nuclear proteins UV cross-linked to CRS RNAs with murine monoclonal antibodies, we now show that p40CRS is heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and p60CRS is polypyrimidine tract-binding protein or hnRNP I. These immunoprecipitation results were confirmed by an immunobinding assay with hnRNP I and hnRNP AI antibodies and by cross-competition electrophoretic mobility shift experiments. In addition, we mapped a putative hnRNP A1 binding site in U5 RNA and demonstrated that p40CRS (hnRNP A1) binding to that site correlates with CRS function. Since both hnRNP I and hnRNP A1 have been shown to influence splicing and potentially other steps in RNA processing, the binding of both hnRNP I and hnRNP A1 to HTLV RNA regulatory elements may alter retrovirus RNA processing and may be involved in regulation by Rex.
PMCID: PMC189599  PMID: 7474099
15.  Polypyrimidine Tract Binding Protein Blocks the 5′ Splice Site Dependent Assembly of U2AF and the Prespliceosomal E Complex 
Molecular cell  2005;19(4):485-496.
Polypyrimidine tract binding protein (PTB) represses the splicing of many alternatively spliced exons. This repression can sometimes involve the direct occlusion of splice sites by PTB. We show here that PTB prevents splicing of the c-src N1 exon to downstream exon 4 by a different mechanism. PTB does not interfere with U1 snRNP binding to the N1 5′ splice site, but instead prevents formation of the pre-spliceosomal Early (E) complex across the intervening intron. If only the repressed 5′ splice site of the N1 exon is present, the splicing factor U2AF does not assemble on the downstream 3′ splice site of exon 4. When the unregulated 5′ splice site of the upstream exon 3 is included in the RNA, U2AF binding is restored and splicing between exons 3 and 4 proceeds, in spite of the presence of the PTB bound across the N1 exon. Rather than directly blocking the N1 splice sites, PTB is blocking the 5′ splice site dependent assembly of U2AF into the E complex, presumably through an interaction with the U1 snRNP. This mechanism of repression is likely to also occur in many other alternative exons.
PMCID: PMC1635971  PMID: 16109373
16.  The Exon Splicing Silencer in Human Immunodeficiency Virus Type 1 Tat Exon 3 Is Bipartite and Acts Early in Spliceosome Assembly 
Molecular and Cellular Biology  1998;18(9):5404-5413.
Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RNA is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals within the HIV-1 genome that control the rate of splicing include weak 3′ splice sites, exon splicing enhancers (ESE), and exon splicing silencers (ESS). We have previously shown that an ESS present within tat exon 2 (ESS2) and a suboptimal 3′ splice site together act to inhibit splicing at the 3′ splice site flanking tat exon 2. This occurs at an early step in spliceosome assembly. Splicing at the 3′ splice site flanking tat exon 3 is regulated by a bipartite element composed of an ESE and an ESS (ESS3). Here we show that ESS3 is composed of two smaller elements (AGAUCC and UUAG) that can inhibit splicing independently. We also show that ESS3 is more active in the context of a heterologous suboptimal splice site than of an optimal 3′ splice site. ESS3 inhibits splicing by blocking the formation of a functional spliceosome at an early step, since A complexes are not detected in the presence of ESS3. Competitor RNAs containing either ESS2 or ESS3 relieve inhibition of splicing of substrates containing ESS3 or ESS2. This suggests that a common cellular factor(s) may be required for the inhibition of tat mRNA splicing mediated by ESS2 and ESS3.
PMCID: PMC109125  PMID: 9710624
17.  Selection of Alternative 5′ Splice Sites: Role of U1 snRNP and Models for the Antagonistic Effects of SF2/ASF and hnRNP A1 
Molecular and Cellular Biology  2000;20(22):8303-8318.
The first component known to recognize and discriminate among potential 5′ splice sites (5′SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5′SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5′SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5′SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5′SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5′SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5′SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.
PMCID: PMC102138  PMID: 11046128
18.  The Germ Cell Nuclear Proteins hnRNP G-T and RBMY Activate a Testis-Specific Exon 
PLoS Genetics  2009;5(11):e1000707.
The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2β is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5′ splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2β and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3′ splice site normally weakly selected in the testis. Co-expression of Tra2β with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein.
Author Summary
This study investigates tissue-specific alternative splicing, which plays a key role in generating diversity in animal cells. We found a new testis-specific exon in a human homologue of the important Drosophila developmental regulator Groucho, which is activated by germ cell RNA binding proteins. By analyzing splicing control of this exon, we elucidated how variations in the activity and expression of splicing regulators together counterbalance splicing activation, and achieve more tightly regulated physiological splicing patterns. We find that although this new human testis-specific exon is not conserved in mice, it is functionally important in that it encodes a peptide which increases the activity of this developmental regulator as a transcriptional repressor. This study provides new insights into how signalling pathways are evolving in human germ cells and the possible molecular defects that might be occurring in infertile men who have genetic deletions of germ cell-specific RNA binding proteins.
PMCID: PMC2762042  PMID: 19893608
19.  HnRNP L and HnRNP A1 Induce Extended U1 snRNA Interactions with an Exon to Repress Spliceosome Assembly 
Molecular cell  2013;49(5):972-982.
Pre-mRNA splicing is catalyzed through the activity of the spliceosome, a dynamic enzymatic complex. Forcing aberrant interactions within the spliceosome can reduce splicing efficiency and alter splice site choice; however, it is unknown whether such alterations are naturally exploited mechanisms of splicing regulation. Here we demonstrate that hnRNP L represses CD45 exon 4 by recruiting hnRNP A1 to a sequence upstream of the 5’ splice site. Together, hnRNP L and A1 induce extended contacts between the 5’ splice site-bound U1 snRNA and neighboring exonic sequences which, in turn, inhibit stable association of U6 snRNA and subsequent catalysis. Importantly, analysis of several exons regulated by hnRNP L shows a clear relationship between the potential for binding of hnRNP A1 and U1 snRNA, and the effect of hnRNP L on splicing. Together our results demonstrate conformational perturbations within the spliceosome are a naturally occurring and generalizable mechanism for controlling alternative splicing decisions.
PMCID: PMC3595347  PMID: 23394998
alternative splicing; U1 snRNA; tri-snRNP; spliceosome assembly; hnRNP L; hnRNP A1
20.  Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation 
Nucleic Acids Research  2012;40(16):8059-8071.
The molecular basis of cell signal-regulated alternative splicing at the 3′ splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3′ splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3′ splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3′ splice site usage.
PMCID: PMC3439897  PMID: 22684629
21.  Cellular RNA Binding Proteins NS1-BP and hnRNP K Regulate Influenza A Virus RNA Splicing 
PLoS Pathogens  2013;9(6):e1003460.
Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA3, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.
Author Summary
Influenza A virus is a major human pathogen, which causes approximately 500,000 deaths/year worldwide. In pandemic years, influenza infection can lead to even higher mortality rates, as in 1918, when ∼30–50 million deaths occurred worldwide. In this manuscript, we identified a novel function for the cellular protein termed NS1-BP as a regulator of the influenza A virus life cycle. We found that NS1-BP, together with other host factors, mediates the expression of a key viral protein termed M2. NS1-BP and its interacting partner hnRNP K specifically regulate alternative splicing of the viral M1 mRNA segment, which generates the M2 mRNA that is translated into the essential viral M2 protein. The M2 protein is key for viral uncoating and entry into the host cell cytoplasm. Altogether, inhibition of NS1-BP and hnRNP K functions regulate influenza A virus gene expression and replication. In sum, these studies revealed new functions for the cellular proteins NS1-BP and hnRNP K during viral RNA expression, which facilitate the influenza A virus life cycle.
PMCID: PMC3694860  PMID: 23825951
22.  Interaction between the Negative Regulator of Splicing Element and a 3′ Splice Site: Requirement for U1 Small Nuclear Ribonucleoprotein and the 3′ Splice Site Branch Point/Pyrimidine Tract 
Journal of Virology  1999;73(3):2394-2400.
The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several cis elements that account for the accumulation of large amounts of unspliced RNA for use as gag-pol mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterologous introns in vivo and in vitro. Previous data showed that the splicing factors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 binding and activity, suggesting that these factors are important for function. These observations, and the finding that a large spliceosome-like complex (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5′ splice site. One model to explain NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3′ splice sites. In this study, we provide evidence that the NRS interacts with an adenovirus 3′ splice site. The interaction was dependent on the integrity of the branch point and pyrimidine tract of the 3′ splice site, and it was sensitive to a mutation that was previously shown to abolish U11 snRNP binding and NRS function. However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3′ splice site correlated with U1, not U11, binding. These results show that the NRS can interact with a 3′ splice site and suggest that U1 is of primary importance for NRS splicing inhibition.
PMCID: PMC104485  PMID: 9971823
23.  Roles for SR Proteins and hnRNP A1 in the Regulation of c-src Exon N1 
Molecular and Cellular Biology  2003;23(6):1874-1884.
The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5′ half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.
PMCID: PMC149473  PMID: 12612063
24.  Intronic Binding Sites for hnRNP A/B and hnRNP F/H Proteins Stimulate Pre-mRNA Splicing 
PLoS Biology  2006;4(2):e21.
hnRNP A/B proteins modulate the alternative splicing of several mammalian and viral pre-mRNAs, and are typically viewed as proteins that enforce the activity of splicing silencers. Here we show that intronic hnRNP A/B–binding sites (ABS) can stimulate the in vitro splicing of pre-mRNAs containing artificially enlarged introns. Stimulation of in vitro splicing could also be obtained by providing intronic ABS in trans through the use of antisense oligonucleotides containing a non-hybridizing ABS-carrying tail. ABS-tailed oligonucleotides also improved the in vivo inclusion of an alternative exon flanked by an enlarged intron. Notably, binding sites for hnRNP F/H proteins (FBS) replicate the activity of ABS by improving the splicing of an enlarged intron and by modulating 5′ splice-site selection. One hypothesis formulated to explain these effects is that bound hnRNP proteins self-interact to bring in closer proximity the external pair of splice sites. Consistent with this model, positioning FBS or ABS at both ends of an intron was required to stimulate splicing of some pre-mRNAs. In addition, a computational analysis of the configuration of putative FBS and ABS located at the ends of introns supports the view that these motifs have evolved to support cooperative interactions. Our results document a positive role for the hnRNP A/B and hnRNP F/H proteins in generic splicing, and suggest that these proteins may modulate the conformation of mammalian pre-mRNAs.
Typically viewed as enforcing splicing silencers, hnRNP A/B proteins may facilitate splicing by modulating the conformation of mammalian pre-mRNAs.
PMCID: PMC1326234  PMID: 16396608
25.  A block in mammalian splicing occurring after formation of large complexes containing U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins. 
Molecular and Cellular Biology  1989;9(1):259-267.
The assembly of mammalian pre-mRNAs into large 50S to 60S complexes, or spliceosomes, containing small nuclear ribonucleoproteins (snRNPs) leads to the production of splicing intermediates, 5' exon and lariat-3' exon, and the subsequent production of spliced products. Influenza virus NS1 mRNA, which encodes a virus-specific protein, is spliced in infected cells to form another viral mRNA (the NS2 mRNA), such that the ratio of unspliced to spliced mRNA is 10 to 1. NS1 mRNA was not detectably spliced in vitro with nuclear extracts from uninfected HeLa cells. Surprisingly, despite the almost total absence of splicing intermediates in the in vitro reaction, NS1 mRNA very efficiently formed ATP-dependent 55S complexes. The formation of 55S complexes with NS1 mRNA was compared with that obtained with an adenovirus pre-mRNA (pKT1 transcript) by using partially purified splicing fractions that restricted the splicing of the pKT1 transcript to the production of splicing intermediates. At RNA precursor levels that were considerably below saturation, approximately 10-fold more of the input NS1 mRNA than of the input pKT1 transcript formed 55S complexes at all time points examined. The pKT1 55S complexes contained splicing intermediates, whereas the NS1 55S complexes contained only precursor NS1 mRNA. Biotin-avidin affinity chromatography showed that the 55S complexes formed with either NS1 mRNA or the pKT1 transcript contained the U1, U2, U4, U5, and U6 snRNPs. Consequently, the formation of 55S complexes containing these five snRNPs was not sufficient for the catalysis of the first step of splicing, indicating that some additional step(s) needs to occur subsequent to this binding. These results indicate that the 5' splice site, 3' and branch point of NS1 and mRNA were capable of interacting with the five snRNPs to form 55S complexes, but apparently some other sequence element(s) in NS1 mRNA blocked the resolution of the 55S complexes that leads to the catalysis of splicing. On the basis of our results, we suggest mechanisms by which the splicing of NS1 is controlled in infected cells.
PMCID: PMC362168  PMID: 2522588

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