Limits...
Competition between a noncoding exon and introns: Gomafu contains tandem UACUAAC repeats and associates with splicing factor-1.

Tsuiji H, Yoshimoto R, Hasegawa Y, Furuno M, Yoshida M, Nakagawa S - Genes Cells (2011)

Bottom Line: Unexpectedly, we found that all Gomafu RNA examined shared a distinctive feature: tandem repeats of UACUAAC, a sequence that has been identified as a conserved intron branch point in the yeast Saccharomyces cerevisiae.The tandem UACUAAC Gomafu RNA repeats bind to the SF1 splicing factor with a higher affinity than the divergent branch point sequence in mammals, which affects the kinetics of the splicing reaction in vitro.We propose that the Gomafu RNA regulates splicing efficiency by changing the local concentration of splicing factors within the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Nakagawa Initiative Research Unit, RIKEN Advanced Science Institute, Hirosawa, Wako, Saitama, Japan.

Show MeSH
TACTAAC repeats in Gomafu delay splicing kinetics in vitro. (A, B) In vitro competition experiments using the Gomafu repeat oligonucleotides. A HeLa cell nuclear extract was pre-incubated on ice, with either water or the oligonucleotides (5 pmol/reaction) used in Fig. 2C. After the addition of the IgM pre-mRNA (A) or AdML pre-mRNA (B), the mixture was incubated at 30 °C for the indicated time. The bands for the RNA products are shown schematically at the right. Note that a marked decrease in the spliced product was observed with the IgM pre-mRNA (dashed box) but not with AdML pre-mRNA. (C) Dose-dependent inhibition of IgM pre-mRNA splicing by the Gomafu repeat oligonucleotides. The in vitro splicing reaction was performed in the presence of water or indicated amount of oligonucleotides at 30 °C for 60 min. Gomafu repeat but not control oligonucleotides inhibited the formation of spliced product in a dose-dependent manner (dashed box). Note that lariat intron is stabilized in the presence of higher amount of oligonucleotides, probably due to an inhibition of endogenous nucleases. (D) Analysis of splicing complex formation. Splicing complexes from the same reaction conditions as in ‘A’ were separated on a native 2% agarose gel. Formation of complex B was significantly retarded in the presence of the Gomafu repeat oligonucleotides. (E) Expression of SF1 in HEK293T cells transfected with control or SF1-expressing vector. Total protein from an equivalent number of cells was separated by 8% SDS–PAGE and detected on the Western blot using anti-FLAG, anti-β-actin and anti-SF1 antibody. The numbers below indicate the relative amount of SF1. (F) Neutralization of inhibitory effect of Gomafu repeat oligonucleotides by an excess amount of SF1. HEK293T cell nuclear extract expressing either empty vector or SF1-FLAG protein was pre-incubated on ice with water or the indicated oligonucleotides (5 pmol/reaction). After the addition of the IgM pre-mRNA, the mixture was incubated at 30 °C for 60 min. Exogenous SF1 protein rescues the splicing efficiency of IgM pre-mRNA (dashed box).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3116199&req=5

fig04: TACTAAC repeats in Gomafu delay splicing kinetics in vitro. (A, B) In vitro competition experiments using the Gomafu repeat oligonucleotides. A HeLa cell nuclear extract was pre-incubated on ice, with either water or the oligonucleotides (5 pmol/reaction) used in Fig. 2C. After the addition of the IgM pre-mRNA (A) or AdML pre-mRNA (B), the mixture was incubated at 30 °C for the indicated time. The bands for the RNA products are shown schematically at the right. Note that a marked decrease in the spliced product was observed with the IgM pre-mRNA (dashed box) but not with AdML pre-mRNA. (C) Dose-dependent inhibition of IgM pre-mRNA splicing by the Gomafu repeat oligonucleotides. The in vitro splicing reaction was performed in the presence of water or indicated amount of oligonucleotides at 30 °C for 60 min. Gomafu repeat but not control oligonucleotides inhibited the formation of spliced product in a dose-dependent manner (dashed box). Note that lariat intron is stabilized in the presence of higher amount of oligonucleotides, probably due to an inhibition of endogenous nucleases. (D) Analysis of splicing complex formation. Splicing complexes from the same reaction conditions as in ‘A’ were separated on a native 2% agarose gel. Formation of complex B was significantly retarded in the presence of the Gomafu repeat oligonucleotides. (E) Expression of SF1 in HEK293T cells transfected with control or SF1-expressing vector. Total protein from an equivalent number of cells was separated by 8% SDS–PAGE and detected on the Western blot using anti-FLAG, anti-β-actin and anti-SF1 antibody. The numbers below indicate the relative amount of SF1. (F) Neutralization of inhibitory effect of Gomafu repeat oligonucleotides by an excess amount of SF1. HEK293T cell nuclear extract expressing either empty vector or SF1-FLAG protein was pre-incubated on ice with water or the indicated oligonucleotides (5 pmol/reaction). After the addition of the IgM pre-mRNA, the mixture was incubated at 30 °C for 60 min. Exogenous SF1 protein rescues the splicing efficiency of IgM pre-mRNA (dashed box).

Mentions: Considering the interaction between the Gomafu RNA and SF1, we hypothesized that the Gomafu RNA might regulate splicing by competing locally with the branch point sequences of pre-mRNAs for the splicing factor SF1. To confirm this hypothesis, we examined the effect of UACUAAC tandem repeats in the Gomafu RNA on the splicing reaction and spliceosome formation in vitro. We first used a model pre-mRNA substrate derived from mouse IgM (Watakabe et al. 1993), which possesses a predictably weak branch point with a degenerate sequence (Guth & Valcarcel 2000). As expected, the addition of the Gomafu repeat oligonucleotides when compared with the control oligonucleotides markedly delayed production of the spliced product (Fig. 4A). We then used another pre-mRNA substrate with strong intron consensus sequences derived from adenovirus (Zapp & Berget 1989). In this case, both the control and Gomafu repeat oligonucleotides inhibited the splicing reaction to some extent; however, no differences were found between the two conditions (Fig. 4B). These results were consistent with previous reports showing that BBP and SF1 are not essential for the splicing reaction itself but are required for optimal removal of introns with sub-optimal consensus sequences (Guth & Valcarcel 2000; Rutz & Seraphin 2000; Tanackovic & Kramer 2005). We then examined whether the Gomafu repeat oligonucleotides inhibit IgM pre-mRNA splicing in a dose-dependent manner. In this experiment, the splicing reaction was performed in an increasing amount of oligonucleotides for a fixed time (60 min). As expected, control oligonucleotides did not inhibit pre-mRNA splicing within a range of 1.25–10 pmol/reaction. On the other hand, inhibitory effect was recognizable with Gomafu repeat oligonucleotitdes as little as 2.5 pmol/reaction, which became clearer with an increased amount of the oligonucleotides (Fig. 4C). We also examined the effect of the Gomafu repeat oligonucleotides on the formation of a spliceosome complex using native gels. Although we did not observe obvious delay in the formation of H/E or A complex, the formation of B complex was markedly delayed in the presence of the Gomafu repeat oligonucleotides (Fig. 4D). Finally, we tested whether these inhibitory effects could be neutralized by an excess amount of SF1 using nuclear extracts prepared from HEK293T cells overexpressing SF1 (Fig. 4E, F). The overexpression resulted in approximately 4 times more SF1 compared with the control cells (Fig. 4E). As expected, the inhibitory effect of the Gomafu repeat oligonucleotides was rescued when using nuclear extracts prepared from SF1-overexpressing cells (Fig. 4F). Taken together, these results suggested that Gomafu RNAs potentially affect kinetics of splicing reaction by competing with endogenous introns for the branch point binding protein SF1.


Competition between a noncoding exon and introns: Gomafu contains tandem UACUAAC repeats and associates with splicing factor-1.

Tsuiji H, Yoshimoto R, Hasegawa Y, Furuno M, Yoshida M, Nakagawa S - Genes Cells (2011)

TACTAAC repeats in Gomafu delay splicing kinetics in vitro. (A, B) In vitro competition experiments using the Gomafu repeat oligonucleotides. A HeLa cell nuclear extract was pre-incubated on ice, with either water or the oligonucleotides (5 pmol/reaction) used in Fig. 2C. After the addition of the IgM pre-mRNA (A) or AdML pre-mRNA (B), the mixture was incubated at 30 °C for the indicated time. The bands for the RNA products are shown schematically at the right. Note that a marked decrease in the spliced product was observed with the IgM pre-mRNA (dashed box) but not with AdML pre-mRNA. (C) Dose-dependent inhibition of IgM pre-mRNA splicing by the Gomafu repeat oligonucleotides. The in vitro splicing reaction was performed in the presence of water or indicated amount of oligonucleotides at 30 °C for 60 min. Gomafu repeat but not control oligonucleotides inhibited the formation of spliced product in a dose-dependent manner (dashed box). Note that lariat intron is stabilized in the presence of higher amount of oligonucleotides, probably due to an inhibition of endogenous nucleases. (D) Analysis of splicing complex formation. Splicing complexes from the same reaction conditions as in ‘A’ were separated on a native 2% agarose gel. Formation of complex B was significantly retarded in the presence of the Gomafu repeat oligonucleotides. (E) Expression of SF1 in HEK293T cells transfected with control or SF1-expressing vector. Total protein from an equivalent number of cells was separated by 8% SDS–PAGE and detected on the Western blot using anti-FLAG, anti-β-actin and anti-SF1 antibody. The numbers below indicate the relative amount of SF1. (F) Neutralization of inhibitory effect of Gomafu repeat oligonucleotides by an excess amount of SF1. HEK293T cell nuclear extract expressing either empty vector or SF1-FLAG protein was pre-incubated on ice with water or the indicated oligonucleotides (5 pmol/reaction). After the addition of the IgM pre-mRNA, the mixture was incubated at 30 °C for 60 min. Exogenous SF1 protein rescues the splicing efficiency of IgM pre-mRNA (dashed box).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3116199&req=5

fig04: TACTAAC repeats in Gomafu delay splicing kinetics in vitro. (A, B) In vitro competition experiments using the Gomafu repeat oligonucleotides. A HeLa cell nuclear extract was pre-incubated on ice, with either water or the oligonucleotides (5 pmol/reaction) used in Fig. 2C. After the addition of the IgM pre-mRNA (A) or AdML pre-mRNA (B), the mixture was incubated at 30 °C for the indicated time. The bands for the RNA products are shown schematically at the right. Note that a marked decrease in the spliced product was observed with the IgM pre-mRNA (dashed box) but not with AdML pre-mRNA. (C) Dose-dependent inhibition of IgM pre-mRNA splicing by the Gomafu repeat oligonucleotides. The in vitro splicing reaction was performed in the presence of water or indicated amount of oligonucleotides at 30 °C for 60 min. Gomafu repeat but not control oligonucleotides inhibited the formation of spliced product in a dose-dependent manner (dashed box). Note that lariat intron is stabilized in the presence of higher amount of oligonucleotides, probably due to an inhibition of endogenous nucleases. (D) Analysis of splicing complex formation. Splicing complexes from the same reaction conditions as in ‘A’ were separated on a native 2% agarose gel. Formation of complex B was significantly retarded in the presence of the Gomafu repeat oligonucleotides. (E) Expression of SF1 in HEK293T cells transfected with control or SF1-expressing vector. Total protein from an equivalent number of cells was separated by 8% SDS–PAGE and detected on the Western blot using anti-FLAG, anti-β-actin and anti-SF1 antibody. The numbers below indicate the relative amount of SF1. (F) Neutralization of inhibitory effect of Gomafu repeat oligonucleotides by an excess amount of SF1. HEK293T cell nuclear extract expressing either empty vector or SF1-FLAG protein was pre-incubated on ice with water or the indicated oligonucleotides (5 pmol/reaction). After the addition of the IgM pre-mRNA, the mixture was incubated at 30 °C for 60 min. Exogenous SF1 protein rescues the splicing efficiency of IgM pre-mRNA (dashed box).
Mentions: Considering the interaction between the Gomafu RNA and SF1, we hypothesized that the Gomafu RNA might regulate splicing by competing locally with the branch point sequences of pre-mRNAs for the splicing factor SF1. To confirm this hypothesis, we examined the effect of UACUAAC tandem repeats in the Gomafu RNA on the splicing reaction and spliceosome formation in vitro. We first used a model pre-mRNA substrate derived from mouse IgM (Watakabe et al. 1993), which possesses a predictably weak branch point with a degenerate sequence (Guth & Valcarcel 2000). As expected, the addition of the Gomafu repeat oligonucleotides when compared with the control oligonucleotides markedly delayed production of the spliced product (Fig. 4A). We then used another pre-mRNA substrate with strong intron consensus sequences derived from adenovirus (Zapp & Berget 1989). In this case, both the control and Gomafu repeat oligonucleotides inhibited the splicing reaction to some extent; however, no differences were found between the two conditions (Fig. 4B). These results were consistent with previous reports showing that BBP and SF1 are not essential for the splicing reaction itself but are required for optimal removal of introns with sub-optimal consensus sequences (Guth & Valcarcel 2000; Rutz & Seraphin 2000; Tanackovic & Kramer 2005). We then examined whether the Gomafu repeat oligonucleotides inhibit IgM pre-mRNA splicing in a dose-dependent manner. In this experiment, the splicing reaction was performed in an increasing amount of oligonucleotides for a fixed time (60 min). As expected, control oligonucleotides did not inhibit pre-mRNA splicing within a range of 1.25–10 pmol/reaction. On the other hand, inhibitory effect was recognizable with Gomafu repeat oligonucleotitdes as little as 2.5 pmol/reaction, which became clearer with an increased amount of the oligonucleotides (Fig. 4C). We also examined the effect of the Gomafu repeat oligonucleotides on the formation of a spliceosome complex using native gels. Although we did not observe obvious delay in the formation of H/E or A complex, the formation of B complex was markedly delayed in the presence of the Gomafu repeat oligonucleotides (Fig. 4D). Finally, we tested whether these inhibitory effects could be neutralized by an excess amount of SF1 using nuclear extracts prepared from HEK293T cells overexpressing SF1 (Fig. 4E, F). The overexpression resulted in approximately 4 times more SF1 compared with the control cells (Fig. 4E). As expected, the inhibitory effect of the Gomafu repeat oligonucleotides was rescued when using nuclear extracts prepared from SF1-overexpressing cells (Fig. 4F). Taken together, these results suggested that Gomafu RNAs potentially affect kinetics of splicing reaction by competing with endogenous introns for the branch point binding protein SF1.

Bottom Line: Unexpectedly, we found that all Gomafu RNA examined shared a distinctive feature: tandem repeats of UACUAAC, a sequence that has been identified as a conserved intron branch point in the yeast Saccharomyces cerevisiae.The tandem UACUAAC Gomafu RNA repeats bind to the SF1 splicing factor with a higher affinity than the divergent branch point sequence in mammals, which affects the kinetics of the splicing reaction in vitro.We propose that the Gomafu RNA regulates splicing efficiency by changing the local concentration of splicing factors within the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Nakagawa Initiative Research Unit, RIKEN Advanced Science Institute, Hirosawa, Wako, Saitama, Japan.

Show MeSH