Ribosomal Protein Binding with the First Intron of the Human rpS26 Pre-mRNA Stimulates Its Interaction with Proteins Extracted from HeLa Cells

As shown by nitrocellulose filtration assays with RNA fragments transcribed from various regions of the human ribosomal protein (rp) S26 gene, proteins of the 40S ribosome subunit bind to the first intron of the rpS26 pre-mRNA. The binding involved mostly S23, S26 and, to a lesser extent, S13/16. Negligible binding was observed for S2/3a, S6, S8, S10, S11, and S20. Small-subunit proteins did not affect the efficiency of in vitro splicing of a pre-mRNA fragment corresponding to the first intron, second exon, second intron, and a part of the third exon of the rpS26 gene. However, ribosomal proteins substantially increased UV-induced adduction of the pre-mRNA fragments with nuclear extract proteins of HeLa cells. The same set of HeLa proteins was observed with each pre-mRNA fragment. Ribosomal proteins formed adducts only in the absence of HeLa proteins.     

Interaction of human S26 ribosomal protein with fragments of mRNA and pre-mRNA for this protein in Hela nuclear cell extracts

As shown by nitrocellulose filtration assays with RNA fragments transcribed from various regions of the human ribosomal protein (rp) S26 gene, recombinant rpS26 binds to the first intron of the rpS26 pre-mRNA (apparent association constant (Ka) approximately 5.0 x 10(7) M-1) and, to a lesser extent, to the rpS26 mRNA (Ka approximately 2.0 x 10(7) M-1). The binding was specific, since human rpS19 had an order of magnitude lower Ka with the first intron and did not bind with the rpS26 mRNA. Immunoassays with specific antibodies showed that rpS26 contained in the nuclear extract of HeLa cells binds to the first intron of its pre-mRNA and, less efficiently, to its mRNA. In either case, RNA binding substantially increased in the presence of recombinant rpS26. Along with other (48 K, 59 K) nuclear proteins, rpS26 was assumed to form complexes, the functional role of which is storage of pre-mRNAs inactive in splicing.     

Human ribosomal protein S13 inhibits splicing of its own pre-mRNA

Recombinant human ribosomal protein (rp) S13 was shown to specifically bind with its own pre-mRNA fragment containing the first exon, first intron, second exon, and a part of the second intron and to inhibit its splicing in vitro. The binding of rpS13 was specific: recombinant human rpS10 and rpS16 bound with the fragment to a lower extent. Moreover, rpS13 binding with the rpS13 pre-mRNA fragment was inhibited by non-labeled poly(AU) and an adenovirus pre-mRNA fragment to a lower extent than by the nonlabeled rpS13 pre-mRNA fragment. The specificity of splicing inhibition was inferred from the finding that, in contrast to rpS13, recombinant rpS10 and rpS16 did not affect the efficiency of first intron excision from the rpS13 pre-mRNA fragment. Enzymatic footprinting was used to determine the rpS13 pre-mRNA nucleotides whose accessibility to RNases T1, T2, and V1 changed in the presence of rpS13. Such nucleotides were detected close to the 5′ and 3′ splicing sites of the first intron. Analysis with the EMBOS-Align program showed that the nucleotide sequence of the first intron of the mammalian rpS13 pre-mRNA is conserved to a greater extent as compared with the other introns. It was assumed that the first intron plays an important role in regulating the expression of the rpS13 gene at the splicing level in all mammals.     

Human ribosomal protein S26 inhibits splicing of its own pre-mRNA

In vitro splicing was studied for a human ribosomal protein (rp) S26 pre-mRNA fragment containing the first exon, first intron, and a part of the second exon. Splicing yielded two products, the first was corresponded to a fragment of the mature rpS26 mRNA and another was retained the 19 3'-terminal nucleotides of the first intron between the first and second exons. Recombinant rpS26 inhibites generation of both splicing products in vitro. The inhibition was specific, because another recombinant human rp, S19, had no effect on the splicing of the pre-mRNA fragment. Toe-printing was used to map the spS26-binding sites of the per-mRNA within the regions of the conventional and alternative 3' splicing sites of the first intron. On the strength of the rusults, rpS26 was assumed to regulate the expression of its own gene at the level of pre-mRNA splicing via a feedback mechanism.     

Human ribosomal protein S13 inhibits splicing of the own pre-mRNA

Recombinant human ribosomal protein S13 (rpS 13) is shown to bind specifically a fragment of its own pre-mRNA that includes exons 1 and 2, intron 1, and part of intron 2, and to inhibit the splicing of that fragment in vitro. The weaker binding of other recombinant human ribosomal proteins, S10 and S16, to this pre-mRNA fragment indicated that the binding of rpS 13 was specific. Besides, poly(AU) and adenovirus pre-mRNA fragment affected poorly the binding of rpS 13 to S13 pre-mRNA, providing another evidence that the interaction was specific. RpS 13 specifically inhibited the pre-mRNA splicing whereas recombinant rpS10 and rpS16 did not affect excision of intron from S13 pre-mRNA fragment in contrast to rpS 13. Those positions in S13 pre-mRNA that were protected by rpS13 protein against cleavage by RNases T1, T2 and V1 were found to be located closely to the 5' and 3' splice sites in the pre-mRNA. Intron 1 in S13 pre-mRNA is more highly conserved within mammals than the other introns in S13 pre-mRNA, which supports the possibility of an important role for intron 1 in the regulation of expression of rpS13 gene in mammals.     

Human Ribosomal Protein S26 Inhibits Splicing of Its Own Pre-mRNA

In vitro splicing was studied for a human ribosomal protein (rp) S26 pre-mRNA fragment containing the first exon, first intron, and a part of the second exon. Splicing yielded two products, one corresponding to a fragment of the mature rpS26 mRNA and the other retaining the 19 3-terminal nucleotides of the first intron between the first and second exons. Recombinant rpS26 inhibited generation of both splicing products in vitro. The inhibition was specific, because another recombinant human rp, S19, had no effect on the splicing of the pre-mRNA fragment. Toe-printing was used to map the rpS26-binding sites of the pre-mRNA in the regions of the conventional and alternative 3 splicing sites of the first intron. On the strength of the results, rpS26 was assumed to regulate the expression of its own gene at the level of pre-mRNA splicing via a feedback mechanism.     

Human ribosomal protein S16 inhibits excision of the first intron from its own pre-mRNA

Recombinant human ribosomal protein S16 (rpS16) is shown to bind specifically to a fragment of its own pre-mRNA that includes exons 1 and 2, intron 1, and part of intron 2, and to inhibit the splicing of the fragment in vitro. The weaker binding of other recombinant human ribosomal proteins, S10 and S13, to this pre-mRNA fragment indicated that the binding of rpS16 was specific. Besides, the poly(AU) and rpS16 mRNA fragment insignificantly affected the binding of rpS16 to its pre-mRNA, providing another evidence that the interaction was specific. rpS16 specifically inhibited the splicing of the pre-mRNA fragment, whereas recombinant rpS10 and rpS13 did not affect intron excision from this pre-mRNA fragment in contrast to rpS16. Those positions in rpS16 pre-mRNA fragment that were protected by rpS16 from cleavage by RNases T1, T2, and V1 were found to be located closely to the branch point and 3’ splice site in the pre-mRNA. The obtained results suggest the possibility of the autoregulation of rpS13 pre-mRNA splicing through the feedback mechanism.     

Interaction of Human Ribosomal Protein S26 with Fragments of Its Own mRNA and Pre-mRNA in Nuclear Extract of HeLa Cells

As shown by nitrocellulose filtration assays with RNA fragments transcribed from various regions of the human ribosomal protein (rp) S26 gene, recombinant rpS26 binds to the first intron of the rpS26 pre-mRNA (apparent association constant (K _a) 5.0 · 10⁷ M^–1) and, to a lesser extent, to the rpS26 mRNA (K _a 2.0 · 10⁷ M^–1). The binding was specific, since human rpS19 had an order of magnitude lower K _a with the first intron and did not bind with the rpS26 mRNA. Immunoassays with specific antibodies showed that rpS26 contained in the nuclear extract of HeLa cells binds to the first intron of its pre-mRNA and, less efficiently, to its mRNA. In either case, RNA binding substantially increased in the presence of recombinant rpS26. Along with other (48K, 59K) nuclear proteins, rpS26 was assumed to form complexes, the functional role of which is storage of pre-mRNAs inactive in splicing.     

Human ribosomal protein S13 regulates expression of its own gene at the splicing step by a feedback mechanism

The expression of ribosomal protein (rp) genes is regulated at multiple levels. In yeast, two genes are autoregulated by feedback effects of the protein on pre-mRNA splicing. Here, we have investigated whether similar mechanisms occur in eukaryotes with more complicated and highly regulated splicing patterns. Comparisons of the sequences of ribosomal protein S13 gene (RPS13) among mammals and birds revealed that intron 1 is more conserved than the other introns. Transfection of HEK 293 cells with a minigene-expressing ribosomal protein S13 showed that the presence of intron 1 reduced expression by a factor of four. Ribosomal protein S13 was found to inhibit excision of intron 1 from rpS13 pre-mRNA fragment in vitro. This protein was shown to be able to specifically bind the fragment and to confer protection against ribonuclease cleavage at sequences near the 5′ and 3′ splice sites. The results suggest that overproduction of rpS13 in mammalian cells interferes with splicing of its own pre-mRNA by a feedback mechanism.     

Polypyrimidine tract binding protein interacts with sequences involved in alternative splicing of beta-tropomyosin pre-mRNA.

Previous studies of alternative splicing of the rat beta-tropomyosin gene have shown that nonmuscle cells contain factors that block the use of the skeletal muscle exon 7 (Guo, W., Mulligan, G. J., Wormsley, S., and Helfman, D. M. (1991) Genes & Dev. 5, 2095-2106). Using an RNA mobility-shift assay we have identified factors in HeLa cell nuclear extracts that specifically interact with sequences responsible for exon blockage. Here we present the purification to apparent homogeneity of a protein that exhibits these sequence specific RNA binding properties. This protein is identical to the polypyrimidine tract binding protein (PTB) which other studies have suggested is involved in the recognition and efficient use of 3'-splice sites. PTB binds to two distinct functional elements within intron 6 of the beta-tropomyosin pre-mRNA: 1) the polypyrimidine tract sequences required for the use of branch points associated with the splicing of exon 7, and 2) the intron regulatory element that is involved in the repression of exon 7. Our results demonstrate that the sequence requirements for PTB binding are different than previously reported and shows that PTB binding cannot be predicted solely on the basis of pyrimidine content. In addition, PTB fails to bind stably to sequences within intron 5 and intron 7 of beta-TM pre-mRNA, yet forms a stable complex with sequences in intron 6, which is not normally spliced in HeLa cells in vitro and in vivo. The nature of the interactions of PTB within this regulated intron reveals several new details about the binding specificity of PTB and suggests that PTB does not function exclusively in a positive manner in the recognition and use of 3'-splice sites.     

A central fragment of ribosomal protein S26 containing the eukaryote-specific motif YxxPKxYxK is a key component of the ribosomal binding site of mRNA region 5' of the E site codon

The eukaryotic ribosomal protein S26e (rpS26e) lacking eubacterial counterparts is a key component of the ribosomal binding site of mRNA region 5' of the codon positioned at the exit site. Here, we determined the rpS26e oligopeptide neighboring mRNA on the human 80S ribosome using mRNA analogues bearing perfluorophenyl azide-derivatized nucleotides at designed locations. The protein was cross-linked to mRNA analogues in specific ribosomal complexes, in which the derivatized nucleotide was located at positions -3 to -9. Digestion of cross-linked rpS26e with various specific proteolytic agents followed by identification of the resulting modified oligopeptides made it possible to map the cross-links to fragment 60-71. This fragment contains the motif YxxPKxYxK conserved in eukaryotic but not in archaeal rpS26e. Analysis of X-ray structure of the Tetrahymena thermophila 40S subunit showed that this motif is not implicated in the intraribosomal interactions, implying its involvement in translation process in a eukaryote-specific manner. Comparison of the results obtained with data on positioning of ribosomal ligands on the 40S subunit lead us to suggest that this motif is involved in interaction with both the 5'-untranslated region of mRNA and the initiation factor eIF3 specific for eukaryotes, providing new insights into molecular mechanisms of translation in eukaryotes.     

Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8

Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26 nt RNA from 5′ end of exon 8, a 33 nt RNA from 3′ end of exon 10 and a 2225 nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.