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Identification of motifs that function in the splicing of non-canonical introns.

Murray JI, Voelker RB, Henscheid KL, Warf MB, Berglund JA - Genome Biol. (2008)

Bottom Line: While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors.In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts.The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.

ABSTRACT

Background: While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors. The diversity of human intronic sequences suggests the existence of novel recognition pathways for non-canonical introns. This study addresses the recognition and splicing of human introns that lack a canonical PY tract. The PY tract is a uridine-rich region at the 3' end of introns that acts as a binding site for U2AF65, a key factor in splicing machinery recruitment.

Results: Human introns were classified computationally into low- and high-scoring PY tracts by scoring the likely U2AF65 binding site strength. Biochemical studies confirmed that low-scoring PY tracts are weak U2AF65 binding sites while high-scoring PY tracts are strong U2AF65 binding sites. A large population of human introns contains weak PY tracts. Computational analysis revealed many families of motifs, including C-rich and G-rich motifs, that are enriched upstream of weak PY tracts. In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts.

Conclusion: The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif.

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ISEs compensate for a weakened PY tract. The four factors present in the early (E) complex (U1 snRNP, SF1, U2AF65 and U2AF35) recognize the four canonical intronic splicing elements (the 5' splice site, the branchpoint (BPS), the PY tract and the 3' splice site). During A complex formation, which follows E complex, the U2 snRNP is recruited by U2AF65 and replaces SF1 at the branchpoint. There are presumably multiple redundant pathways that compensate for weak U2AF65-PY tract interactions, including bridging interactions between SF1, U2AF65 and U2AF35, alternative PY tract binding proteins (shown here as factor 'P'), and pathways involving additional non-canonical motifs such as ESEs or ISEs. We propose that ISEs in the region upstream of a weak PY tract (nucleotides -30 to -80) are important for recognizing introns with weak PY tracts. Specifically, we have shown that G-rich and C-rich motifs are ISEs that compensate for weakened U2AF65-PY tract interactions. Factors X and Y represent proteins binding the compensating ISEs. We propose that ISE-factor X/Y interactions can compensate for weak PY tract-U2AF65 interactions and help recruit the U2 snRNP to the branchpoint. The dash (//) indicates the variable length between the 5' splice site and 3' end of the intron.
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Figure 10: ISEs compensate for a weakened PY tract. The four factors present in the early (E) complex (U1 snRNP, SF1, U2AF65 and U2AF35) recognize the four canonical intronic splicing elements (the 5' splice site, the branchpoint (BPS), the PY tract and the 3' splice site). During A complex formation, which follows E complex, the U2 snRNP is recruited by U2AF65 and replaces SF1 at the branchpoint. There are presumably multiple redundant pathways that compensate for weak U2AF65-PY tract interactions, including bridging interactions between SF1, U2AF65 and U2AF35, alternative PY tract binding proteins (shown here as factor 'P'), and pathways involving additional non-canonical motifs such as ESEs or ISEs. We propose that ISEs in the region upstream of a weak PY tract (nucleotides -30 to -80) are important for recognizing introns with weak PY tracts. Specifically, we have shown that G-rich and C-rich motifs are ISEs that compensate for weakened U2AF65-PY tract interactions. Factors X and Y represent proteins binding the compensating ISEs. We propose that ISE-factor X/Y interactions can compensate for weak PY tract-U2AF65 interactions and help recruit the U2 snRNP to the branchpoint. The dash (//) indicates the variable length between the 5' splice site and 3' end of the intron.

Mentions: Our results suggest a model where ISEs present upstream of a weak PY tract compensate for a weakened U2AF65-RNA interaction (Figure 10). In the case of LCAT intron 4, the G-rich and C-rich motifs and the branchpoint sequence are highly conserved and yet the PY tract is not well conserved. It is possible that the presence of strong enhancers upstream of the PY tract has allowed for greater degeneracy in the PY tract region. In support of this model we have observed that when the PY tract is strengthened to include a run of eight uridines, mutation of both C-rich motifs or the cumulative mutation of the G-rich and C-rich motifs no longer have an effect on LCAT intron 4 splicing (Figure 8). The G-rich and C-rich motifs appear dispensable in the presence of a strong PY tract. G-rich motifs have previously been shown to be dispensable for maximal splicing in the presence of a strengthened PY tract [20]. These results suggest that strong U2AF65-PY tract interactions alleviate the role of upstream ISEs.


Identification of motifs that function in the splicing of non-canonical introns.

Murray JI, Voelker RB, Henscheid KL, Warf MB, Berglund JA - Genome Biol. (2008)

ISEs compensate for a weakened PY tract. The four factors present in the early (E) complex (U1 snRNP, SF1, U2AF65 and U2AF35) recognize the four canonical intronic splicing elements (the 5' splice site, the branchpoint (BPS), the PY tract and the 3' splice site). During A complex formation, which follows E complex, the U2 snRNP is recruited by U2AF65 and replaces SF1 at the branchpoint. There are presumably multiple redundant pathways that compensate for weak U2AF65-PY tract interactions, including bridging interactions between SF1, U2AF65 and U2AF35, alternative PY tract binding proteins (shown here as factor 'P'), and pathways involving additional non-canonical motifs such as ESEs or ISEs. We propose that ISEs in the region upstream of a weak PY tract (nucleotides -30 to -80) are important for recognizing introns with weak PY tracts. Specifically, we have shown that G-rich and C-rich motifs are ISEs that compensate for weakened U2AF65-PY tract interactions. Factors X and Y represent proteins binding the compensating ISEs. We propose that ISE-factor X/Y interactions can compensate for weak PY tract-U2AF65 interactions and help recruit the U2 snRNP to the branchpoint. The dash (//) indicates the variable length between the 5' splice site and 3' end of the intron.
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Figure 10: ISEs compensate for a weakened PY tract. The four factors present in the early (E) complex (U1 snRNP, SF1, U2AF65 and U2AF35) recognize the four canonical intronic splicing elements (the 5' splice site, the branchpoint (BPS), the PY tract and the 3' splice site). During A complex formation, which follows E complex, the U2 snRNP is recruited by U2AF65 and replaces SF1 at the branchpoint. There are presumably multiple redundant pathways that compensate for weak U2AF65-PY tract interactions, including bridging interactions between SF1, U2AF65 and U2AF35, alternative PY tract binding proteins (shown here as factor 'P'), and pathways involving additional non-canonical motifs such as ESEs or ISEs. We propose that ISEs in the region upstream of a weak PY tract (nucleotides -30 to -80) are important for recognizing introns with weak PY tracts. Specifically, we have shown that G-rich and C-rich motifs are ISEs that compensate for weakened U2AF65-PY tract interactions. Factors X and Y represent proteins binding the compensating ISEs. We propose that ISE-factor X/Y interactions can compensate for weak PY tract-U2AF65 interactions and help recruit the U2 snRNP to the branchpoint. The dash (//) indicates the variable length between the 5' splice site and 3' end of the intron.
Mentions: Our results suggest a model where ISEs present upstream of a weak PY tract compensate for a weakened U2AF65-RNA interaction (Figure 10). In the case of LCAT intron 4, the G-rich and C-rich motifs and the branchpoint sequence are highly conserved and yet the PY tract is not well conserved. It is possible that the presence of strong enhancers upstream of the PY tract has allowed for greater degeneracy in the PY tract region. In support of this model we have observed that when the PY tract is strengthened to include a run of eight uridines, mutation of both C-rich motifs or the cumulative mutation of the G-rich and C-rich motifs no longer have an effect on LCAT intron 4 splicing (Figure 8). The G-rich and C-rich motifs appear dispensable in the presence of a strong PY tract. G-rich motifs have previously been shown to be dispensable for maximal splicing in the presence of a strengthened PY tract [20]. These results suggest that strong U2AF65-PY tract interactions alleviate the role of upstream ISEs.

Bottom Line: While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors.In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts.The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.

ABSTRACT

Background: While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors. The diversity of human intronic sequences suggests the existence of novel recognition pathways for non-canonical introns. This study addresses the recognition and splicing of human introns that lack a canonical PY tract. The PY tract is a uridine-rich region at the 3' end of introns that acts as a binding site for U2AF65, a key factor in splicing machinery recruitment.

Results: Human introns were classified computationally into low- and high-scoring PY tracts by scoring the likely U2AF65 binding site strength. Biochemical studies confirmed that low-scoring PY tracts are weak U2AF65 binding sites while high-scoring PY tracts are strong U2AF65 binding sites. A large population of human introns contains weak PY tracts. Computational analysis revealed many families of motifs, including C-rich and G-rich motifs, that are enriched upstream of weak PY tracts. In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts.

Conclusion: The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif.

Show MeSH