scholarly journals Multiple Roles for SR Proteins in trans Splicing

2002 ◽  
Vol 22 (15) ◽  
pp. 5337-5346 ◽  
Author(s):  
Suzanne Furuyama ◽  
James P. Bruzik
Keyword(s):  
Splice Site ◽  
Rna Binding ◽  
Sr Proteins ◽  
Multiple Roles ◽  
Separate Experiment ◽  
Splice Sites ◽  
U2 Snrnp ◽  
In Trans ◽  
Two Stages

ABSTRACT The trans-splicing reaction involves the association of 5′; and 3′; splice sites contained on separate transcripts. The mechanism by which these splice sites are juxtaposed during trans-spliceosome assembly and the role of SR proteins at each stage in this process have not been determined. Utilizing a system that allows for the separation of the RNA binding and RS domains of SR proteins, we have found that SR proteins are required for at least two stages of the trans-splicing reaction. They are important both prior to and subsequent to the addition of U2 snRNP to the 3′; acceptor. In addition, we have demonstrated a role for RS domain phosphorylation in both of these activities. Dephosphorylation of the RS domain led to a block in U2 snRNP binding to the substrate. In a separate experiment, RS domain phosphorylation was also determined to be necessary for trans splicing to proceed on a substrate that had U2 snRNP already bound. This newly identified role for phosphorylated SR proteins post-U2-snRNP addition coincides with the recruitment of the 5′; splice site contained on the SL RNP, suggesting a role for SR proteins in splice site communication in trans splicing.

scholarly journals Acute depletion of METTL3 identifies a role for N6-methyladenosine in alternative intron/exon inclusion in the nascent transcriptome

2020 ◽  
Author(s):  
Guifeng Wei ◽  
Mafalda Almeida ◽  
Greta Pintacuda ◽  
Heather Coker ◽  
Joseph S Bowness ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Histone Modification ◽  
Splice Site ◽  
Rna Processing ◽  
Binding Sites ◽  
Rna Binding ◽  
Embryonic Stem ◽  
Splice Sites ◽  
Rna Regulation

AbstractRNA N6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed co-transcriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells (mESCs). We find that around 10% m6A peaks are in introns, often close to 5’-splice sites. RNA m6A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Interestingly, acute dTAG depletion of METTL3 reveals that inclusion of m6A-bearing alternative introns/exons in the nascent transcriptome is disrupted. For terminal or variable-length exons, m6A peaks are generally located upstream of a repressed 5’-splice site, and downstream of an enhanced 5’-splice site. Intriguingly, genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Our findings demonstrate a direct crosstalk between m6A machinery and the regulation of RNA processing.

scholarly journals Role of the 3′ Splice Site in U12-Dependent Intron Splicing

2001 ◽  
Vol 21 (6) ◽  
pp. 1942-1952 ◽  
Author(s):  
Rosemary C. Dietrich ◽  
Marian J. Peris ◽  
Andrew S. Seyboldt ◽  
Richard A. Padgett
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Intron Splicing ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Branch Site ◽  
Site Function

ABSTRACT U12-dependent introns containing alterations of the 3′ splice site AC dinucleotide or alterations in the spacing between the branch site and the 3′ splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5′ splice site AU dinucleotide, any nucleotide could serve as the 3′-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3′ splice site function. A branch site-to-3′ splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3′ splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3′ splice site but that formation of the prespliceosomal complex A requires an active 3′ splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3′ splice site.

scholarly journals HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo

2017 ◽  
Author(s):  
Jonathan M. Howard ◽  
Hai Lin ◽  
Garam Kim ◽  
Jolene M Draper ◽  
Maximilian Haeussler ◽  
...  
Keyword(s):  
Splice Site ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Regulatory Elements ◽  
Splice Sites ◽  
Spliceosome Assembly ◽  
Over Expression

AbstractAlternative pre-mRNA splicing plays a major role in expanding the transcript output of human genes. This process is regulated, in part, by the interplay of trans-acting RNA binding proteins (RBPs) with myriad cis-regulatory elements scattered throughout pre-mRNAs. These molecular recognition events are critical for defining the protein coding sequences (exons) within pre-mRNAs and directing spliceosome assembly on non-coding regions (introns). One of the earliest events in this process is recognition of the 3’ splice site by U2 small nuclear RNA auxiliary factor 2 (U2AF2). Splicing regulators, such as the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), influence spliceosome assembly both in vitro and in vivo, but their mechanisms of action remain poorly described on a global scale. HNRNPA1 also promotes proof reading of 3’ss sequences though a direct interaction with the U2AF heterodimer. To determine how HNRNPA1 regulates U2AF-RNA interactions in vivo, we analyzed U2AF2 RNA binding specificity using individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) in control- and HNRNPA1 over-expression cells. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3’ splice sites of alternative cassette exons but not constitutive exons upon HNRNPA1 over-expression. A subset of these events shows a concomitant increase of U2AF2 crosslinking at distal intronic regions, suggesting a shift of U2AF2 to “decoy” binding sites. Of the many non-canonical U2AF2 binding sites, Alu-derived RNA sequences represented one of the most abundant classes of HNRNPA1-dependent decoys. Splicing reporter assays demonstrated that mutation of U2AF2 decoy sites inhibited HNRNPA1-dependent exon skipping in vivo. We propose that HNRNPA1 regulates exon definition by modulating the interaction of U2AF2 with decoy or bona fide 3’ splice sites.

SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex

1994 ◽  
Vol 14 (11) ◽  
pp. 7670-7682
Author(s):  
D Staknis ◽  
R Reed
Keyword(s):  
Protein Interactions ◽  
Splice Site ◽  
Sr Proteins ◽  
Cross Linking ◽  
Splice Sites ◽  
Sr Protein ◽  
Specific Recognition ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

We show that addition of SR proteins to in vitro splicing extracts results in a significant increase in assembly of the earliest prespliceosomal complex E and a corresponding decrease in assembly of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex H. In addition, SR proteins promote formation of the E5' and E3' complexes that assemble on RNAs containing only 5' and 3' splice sites, respectively. We conclude that SR proteins promote the earliest specific recognition of both the 5' and 3' splice sites and are limiting for this function in HeLa nuclear extracts. Using UV cross-linking, we demonstrate specific, splice site-dependent RNA-protein interactions of SR proteins in the E, E5', and E3' complexes. SR proteins do not UV cross-link in the H complex, and conversely, hnRNP cross-linking is largely excluded from the E-type complexes. We also show that a discrete complex resembling the E5' complex assembles on both purine-rich and non-purine-rich exonic splicing enhancers. This complex, which we have designated the Enhancer complex, contains U1 small nuclear RNP (snRNP) and is associated with different SR protein family members, depending on the sequence of the enhancer. We propose that both downstream 5' splice site enhancers and exonic enhancers function by establishing a network of pre-mRNA-protein and protein-protein interactions involving U1 snRNP, SR proteins, and U2AF that is similar to the interactions that bring the 5' and 3' splice sites together in the E complex.

scholarly journals The RNA-Binding Protein TIA-1 Is a Novel Mammalian Splicing Regulator Acting through Intron Sequences Adjacent to a 5′ Splice Site

2000 ◽  
Vol 20 (17) ◽  
pp. 6287-6299 ◽  
Author(s):  
Fabienne Del Gatto-Konczak ◽  
Cyril F. Bourgeois ◽  
Caroline Le Guiner ◽  
Liliane Kister ◽  
Marie-Claude Gesnel ◽  
...  
Keyword(s):  
Splice Site ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Dependent Manner ◽  
Splice Sites ◽  
Protein Fusion ◽  
Cell Extracts ◽  
U1 Snrnp ◽  
Splicing Regulator

ABSTRACT Splicing of the K-SAM alternative exon of the fibroblast growth factor receptor 2 gene is heavily dependent on the U-rich sequence IAS1 lying immediately downstream from its 5′ splice site. We show that IAS1 can activate the use of several heterologous 5′ splice sites in vitro. Addition of the RNA-binding protein TIA-1 to splicing extracts preferentially enhances the use of 5′ splice sites linked to IAS1. TIA-1 can provoke a switch to use of such sites on pre-mRNAs with competing 5′ splice sites, only one of which is adjacent to IAS1. Using a combination of UV cross-linking and specific immunoprecipitation steps, we show that TIA-1 binds to IAS1 in cell extracts. This binding is stronger if IAS1 is adjacent to a 5′ splice site and is U1 snRNP dependent. Overexpression of TIA-1 in cultured cells activates K-SAM exon splicing in an IAS1-dependent manner. If IAS1 is replaced with a bacteriophage MS2 operator, splicing of the K-SAM exon can no longer be activated by TIA-1. Splicing can, however, be activated by a TIA-1–MS2 coat protein fusion, provided that the operator is close to the 5′ splice site. Our results identify TIA-1 as a novel splicing regulator, which acts by binding to intron sequences immediately downstream from a 5′ splice site in a U1 snRNP-dependent fashion. TIA-1 is distantly related to the yeast U1 snRNP protein Nam8p, and the functional similarities between the two proteins are discussed.

scholarly journals In Vivo Requirement of the Small Subunit of U2AF for Recognition of a Weak 3′ Splice Site

2006 ◽  
Vol 26 (21) ◽  
pp. 8183-8190 ◽  
Author(s):  
Teresa R. Pacheco ◽  
Miguel B. Coelho ◽  
Joana M. P. Desterro ◽  
Inês Mollet ◽  
Maria Carmo-Fonseca
Keyword(s):  
Splice Site ◽  
Small Subunit ◽  
Splice Sites ◽  
Polypyrimidine Tract ◽  
Interaction Domain ◽  
Specific Subset ◽  
U2 Snrnp ◽  
Selection Of

ABSTRACT The U2 snRNP auxiliary factor (U2AF) is an essential splicing factor composed of two subunits, a large, 65-kDa subunit (U2AF65) and a small subunit, U2AF35. U2AF65 binds to the polypyrimidine tract upstream from the 3′ splice site and promotes U2 snRNP binding to the pre-mRNA. Based on in vitro studies, it has been proposed that U2AF35 plays a role in assisting U2AF65 recruitment to nonconsensus polypyrimidine tracts. Here we have analyzed in vivo the roles of the two subunits of U2AF in the selection between alternative 3′ splice sites associated with polypyrimidine tracts of different strengths. Our results reveal a feedback mechanism by which RNA interference (RNAi)-mediated depletion of U2AF65 triggers the downregulation of U2AF35. We further show that the knockdown of each U2AF subunit inhibits weak 3′ splice site recognition, while overexpression of U2AF65 alone is sufficient to activate the selection of this splice site. A variant of U2AF65 lacking the interaction domain with U2AF35 shows a reduced ability to promote this splicing event, suggesting that recognition of the weak 3′ splice site involves the U2AF heterodimer. Furthermore, our data suggest that, rather than being required for splicing of all pre-mRNA substrates containing a weak polypyrimidine tract, U2AF35 regulates the selection of weak 3′ splice sites in a specific subset of cellular transcripts.

scholarly journals Spliceosome Assembly Pathways for Different Types of Alternative Splicing Converge during Commitment to Splice Site Pairing in the A Complex

2008 ◽  
Vol 29 (4) ◽  
pp. 1072-1082 ◽  
Author(s):  
Matthew V. Kotlajich ◽  
Tara L. Crabb ◽  
Klemens J. Hertel
Keyword(s):  
Alternative Splicing ◽  
Branch Point ◽  
Splice Site ◽  
Atp Hydrolysis ◽  
Splice Sites ◽  
Mrna Isoforms ◽  
Exon Definition ◽  
U2 Snrnp ◽  
Kinetic Trap ◽  
Site Recognition

ABSTRACT Differential splice site pairing establishes alternative splicing patterns resulting in the generation of multiple mRNA isoforms. This process is carried out by the spliceosome, which is activated by a series of sequential structural rearrangements of its five core snRNPs. To determine when splice sites become functionally paired, we carried out a series of kinetic trap experiments using pre-mRNAs that undergo alternative 5′ splice site selection or alternative exon inclusion. We show that commitment to splice site pairing in both cases occurs in the A complex, which is characterized by the ATP-dependent association of the U2 snRNP with the branch point. Interestingly, the timing of splice site pairing is independent of the intron or exon definition modes of splice site recognition. Using the ATP analog ATPγS, we showed that ATP hydrolysis is required for splice site pairing independent from U2 snRNP binding to the pre-mRNA. These results identify the A complex as the spliceosomal assembly step dedicated to splice site pairing and suggest that ATP hydrolysis locks splice sites into a splicing pattern after stable U2 snRNP association to the branch point.

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

1997 ◽  
Vol 138 (2) ◽  
pp. 225-238 ◽  
Author(s):  
Javier F. Cáceres ◽  
Tom Misteli ◽  
Gavin R. Screaton ◽  
David L. Spector ◽  
Adrian R. Krainer
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Site Selection ◽  
Cellular Localization ◽  
Sr Proteins ◽  
Dependent Manner ◽  
Splice Site Selection ◽  
Subnuclear Localization

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.

scholarly journals U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor of 65 kDa, U2AF65, can promote U1 snRNP recruitment to 5′ splice sites

2003 ◽  
Vol 372 (1) ◽  
pp. 235-240 ◽  
Author(s):  
Patrik FÖRCH ◽  
Livia MERENDINO ◽  
Concepción MARTÍNEZ ◽  
Juan VALCÁRCEL
Keyword(s):  
Splice Sites ◽  
Ribonucleoprotein Particle ◽  
Rich Domain ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Nuclear Ribonucleoprotein ◽  
Auxiliary Factor ◽  
Small Nuclear Ribonucleoprotein Particle

The splicing factor U2AF65, U2 small nuclear ribonucleoprotein particle (snRNP) auxillary factor of 65 kDa, binds to pyrimidine-rich sequences at 3′ splice sites to recruit U2 snRNP to pre-mRNAs. We report that U2AF65 can also promote the recruitment of U1 snRNP to weak 5′ splice sites that are followed by uridine-rich sequences. The arginine- and serine-rich domain of U2AF65 is critical for U1 recruitment, and we discuss the role of its RNA–RNA annealing activity in this novel function of U2AF65.

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