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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.

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Nuclear localization signals are redundantly distributed in the Gomafu RNA. (A) Schematic drawing of the Gomafu RNA fragments introduced into the Neuro2A cells. Asterisks show the positions of the seven UACUAAC repeats in the Gomafu RNA. (B) Subcellular localization of the Gomafu RNA fragments revealed by FISH. All of the Gomafu RNA fragments were localized in the nucleus, regardless of the presence of the UACUAAC repeat. (C) Western blot analysis of siRNA-mediated depletion of SF1 in the Neuro2A. The values below indicate the quantification of the western blot signals. (D) Distribution of Gomafu RNA in the SF1-depleted cells. Note that the nuclear localization was not affected by the SF1 knockdown. (E) Schematic drawing of the structure of Slc8a1. Asterisks show the positions of the eight UACUAAC sequences. (F) Localization of Malat1 and Slc8a1 transcripts in adult kidney cortex. The in situ hybridization signals were detected using the NBT/BCIP development method. Note that Slc8a1 transcripts are predominantly distributed in the cytoplasm surrounding the nucleus. Insets show higher magnification images. Scale bars, 10 μm.
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fig03: Nuclear localization signals are redundantly distributed in the Gomafu RNA. (A) Schematic drawing of the Gomafu RNA fragments introduced into the Neuro2A cells. Asterisks show the positions of the seven UACUAAC repeats in the Gomafu RNA. (B) Subcellular localization of the Gomafu RNA fragments revealed by FISH. All of the Gomafu RNA fragments were localized in the nucleus, regardless of the presence of the UACUAAC repeat. (C) Western blot analysis of siRNA-mediated depletion of SF1 in the Neuro2A. The values below indicate the quantification of the western blot signals. (D) Distribution of Gomafu RNA in the SF1-depleted cells. Note that the nuclear localization was not affected by the SF1 knockdown. (E) Schematic drawing of the structure of Slc8a1. Asterisks show the positions of the eight UACUAAC sequences. (F) Localization of Malat1 and Slc8a1 transcripts in adult kidney cortex. The in situ hybridization signals were detected using the NBT/BCIP development method. Note that Slc8a1 transcripts are predominantly distributed in the cytoplasm surrounding the nucleus. Insets show higher magnification images. Scale bars, 10 μm.

Mentions: Because the tandem TACTAAC repeats were the only conserved feature found in the Gomafu homologues in different vertebrate species, we speculated that this sequence might regulate nuclear localization. We therefore stably transfected fragments of Gomafu that did or did not contain the TACTAAC repeats into Neuro2A cells (Fig. 3A, B). All five of the fragment of Gomafu RNA as well as the full-length Gomafu RNA were localized to the nucleus when overexpressed in Neuro2A cells (Fig. 3B), suggesting that the nuclear localization elements are widely distributed throughout the Gomafu RNA and that the repeats are not necessary for the nuclear localization of 5′ and 3′ fragments of Gomafu RNA. We further examined the effect of SF1 knockdown on the Gomafu RNA (Fig. 3C, D) to determine whether SF1 regulates nuclear retention or stability of the Gomafu RNA. The siRNA efficiently depleted SF1 (Fig. 3C); however, stability or subcellular localization of the Gomafu RNA was not significantly influenced (Fig. 3D), suggesting that SF1 might act downstream of the Gomafu RNA rather than regulating its stability or localization. We also examined the expression of Slc8a1, which contains eight tandem TACTAAC repeats (Table 1, Fig. 3E). Unlike nuclear-localizing Malat1 RNA, transcripts of Slc8a1 were predominantly localized to the cytoplasm (Fig. 3F). These signals were not detected with sense probes for Slc8a1 (data not shown). Thus, the presence of multiple UACUAAC sequences was not sufficient for the nuclear retention of mRNA.


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)

Nuclear localization signals are redundantly distributed in the Gomafu RNA. (A) Schematic drawing of the Gomafu RNA fragments introduced into the Neuro2A cells. Asterisks show the positions of the seven UACUAAC repeats in the Gomafu RNA. (B) Subcellular localization of the Gomafu RNA fragments revealed by FISH. All of the Gomafu RNA fragments were localized in the nucleus, regardless of the presence of the UACUAAC repeat. (C) Western blot analysis of siRNA-mediated depletion of SF1 in the Neuro2A. The values below indicate the quantification of the western blot signals. (D) Distribution of Gomafu RNA in the SF1-depleted cells. Note that the nuclear localization was not affected by the SF1 knockdown. (E) Schematic drawing of the structure of Slc8a1. Asterisks show the positions of the eight UACUAAC sequences. (F) Localization of Malat1 and Slc8a1 transcripts in adult kidney cortex. The in situ hybridization signals were detected using the NBT/BCIP development method. Note that Slc8a1 transcripts are predominantly distributed in the cytoplasm surrounding the nucleus. Insets show higher magnification images. Scale bars, 10 μm.
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fig03: Nuclear localization signals are redundantly distributed in the Gomafu RNA. (A) Schematic drawing of the Gomafu RNA fragments introduced into the Neuro2A cells. Asterisks show the positions of the seven UACUAAC repeats in the Gomafu RNA. (B) Subcellular localization of the Gomafu RNA fragments revealed by FISH. All of the Gomafu RNA fragments were localized in the nucleus, regardless of the presence of the UACUAAC repeat. (C) Western blot analysis of siRNA-mediated depletion of SF1 in the Neuro2A. The values below indicate the quantification of the western blot signals. (D) Distribution of Gomafu RNA in the SF1-depleted cells. Note that the nuclear localization was not affected by the SF1 knockdown. (E) Schematic drawing of the structure of Slc8a1. Asterisks show the positions of the eight UACUAAC sequences. (F) Localization of Malat1 and Slc8a1 transcripts in adult kidney cortex. The in situ hybridization signals were detected using the NBT/BCIP development method. Note that Slc8a1 transcripts are predominantly distributed in the cytoplasm surrounding the nucleus. Insets show higher magnification images. Scale bars, 10 μm.
Mentions: Because the tandem TACTAAC repeats were the only conserved feature found in the Gomafu homologues in different vertebrate species, we speculated that this sequence might regulate nuclear localization. We therefore stably transfected fragments of Gomafu that did or did not contain the TACTAAC repeats into Neuro2A cells (Fig. 3A, B). All five of the fragment of Gomafu RNA as well as the full-length Gomafu RNA were localized to the nucleus when overexpressed in Neuro2A cells (Fig. 3B), suggesting that the nuclear localization elements are widely distributed throughout the Gomafu RNA and that the repeats are not necessary for the nuclear localization of 5′ and 3′ fragments of Gomafu RNA. We further examined the effect of SF1 knockdown on the Gomafu RNA (Fig. 3C, D) to determine whether SF1 regulates nuclear retention or stability of the Gomafu RNA. The siRNA efficiently depleted SF1 (Fig. 3C); however, stability or subcellular localization of the Gomafu RNA was not significantly influenced (Fig. 3D), suggesting that SF1 might act downstream of the Gomafu RNA rather than regulating its stability or localization. We also examined the expression of Slc8a1, which contains eight tandem TACTAAC repeats (Table 1, Fig. 3E). Unlike nuclear-localizing Malat1 RNA, transcripts of Slc8a1 were predominantly localized to the cytoplasm (Fig. 3F). These signals were not detected with sense probes for Slc8a1 (data not shown). Thus, the presence of multiple UACUAAC sequences was not sufficient for the nuclear retention of mRNA.

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