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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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G-rich and C-rich motifs function combinatorially in LCAT intron 4 splicing. (a) LCAT intron 4 with the mutations shown in blue above the WT sequence. BPS, branchpoint. (b) Splicing of the LCAT intron 4 mini-genes (WT, MUT1, MUT3, MUT6, MUT 24 and MUT 25) in HeLa cells. Analysis was performed as in Figure 4. (c) Graphical representation of the percent pre-mRNA for each LCAT mini-gene. Error bars represent standard deviation of replicate experiments.
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Figure 6: G-rich and C-rich motifs function combinatorially in LCAT intron 4 splicing. (a) LCAT intron 4 with the mutations shown in blue above the WT sequence. BPS, branchpoint. (b) Splicing of the LCAT intron 4 mini-genes (WT, MUT1, MUT3, MUT6, MUT 24 and MUT 25) in HeLa cells. Analysis was performed as in Figure 4. (c) Graphical representation of the percent pre-mRNA for each LCAT mini-gene. Error bars represent standard deviation of replicate experiments.

Mentions: We hypothesized that the G-rich motifs and C-rich motifs could be functioning together in the recognition of LCAT intron 4. We have observed that there are many examples of introns where the G-rich and C-rich motifs are both present (data not shown). Mutation of both GRM1 and CRM1 (MUT 24, Figure 6a) resulted in a greater decrease in splicing (shown as an increase in percent pre-mRNA) than mutation of either motif alone (Figure 6b, compare MUT 24, lane 5, to MUT 1, lane 2, or MUT 3, lane 3; Figure 6c). An even greater decrease in splicing was observed for the combined mutation of GRM1, CRM1 and CRM2 (MUT 25, Figure 6b, compare MUT 25, lane 6, to MUT 3, lane 3 or MUT 6, lane 4; Figure 6c). These results suggest that the G-rich motifs and C-rich motifs function in combination to promote the splicing of LCAT intron 4.


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)

G-rich and C-rich motifs function combinatorially in LCAT intron 4 splicing. (a) LCAT intron 4 with the mutations shown in blue above the WT sequence. BPS, branchpoint. (b) Splicing of the LCAT intron 4 mini-genes (WT, MUT1, MUT3, MUT6, MUT 24 and MUT 25) in HeLa cells. Analysis was performed as in Figure 4. (c) Graphical representation of the percent pre-mRNA for each LCAT mini-gene. Error bars represent standard deviation of replicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2481429&req=5

Figure 6: G-rich and C-rich motifs function combinatorially in LCAT intron 4 splicing. (a) LCAT intron 4 with the mutations shown in blue above the WT sequence. BPS, branchpoint. (b) Splicing of the LCAT intron 4 mini-genes (WT, MUT1, MUT3, MUT6, MUT 24 and MUT 25) in HeLa cells. Analysis was performed as in Figure 4. (c) Graphical representation of the percent pre-mRNA for each LCAT mini-gene. Error bars represent standard deviation of replicate experiments.
Mentions: We hypothesized that the G-rich motifs and C-rich motifs could be functioning together in the recognition of LCAT intron 4. We have observed that there are many examples of introns where the G-rich and C-rich motifs are both present (data not shown). Mutation of both GRM1 and CRM1 (MUT 24, Figure 6a) resulted in a greater decrease in splicing (shown as an increase in percent pre-mRNA) than mutation of either motif alone (Figure 6b, compare MUT 24, lane 5, to MUT 1, lane 2, or MUT 3, lane 3; Figure 6c). An even greater decrease in splicing was observed for the combined mutation of GRM1, CRM1 and CRM2 (MUT 25, Figure 6b, compare MUT 25, lane 6, to MUT 3, lane 3 or MUT 6, lane 4; Figure 6c). These results suggest that the G-rich motifs and C-rich motifs function in combination to promote the splicing of LCAT intron 4.

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