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 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.     

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.     

Ribosomal protein binding with the first intron of the human ribosomal protein S26 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.     

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.     

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.     

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.     

Characterization of RNA binding protein RBP-P reveals a possible role in rice glutelin gene expression and RNA localization

RNA binding proteins (RBPs) play an important role in mRNA metabolism including synthesis, maturation, transport, localization, and stability. In developing rice seeds, RNAs that code for the major storage proteins are transported to specific domains of the cortical endoplasmic reticulum (ER) by a regulated mechanism requiring RNA cis-localization elements, or zipcodes. Putative trans-acting RBPs that recognize prolamine RNA zipcodes required for restricted localization to protein body-ER have previously been identified. Here, we describe the identification of RBP-P using a Northwestern blot approach as an RBP that recognizes and binds to glutelin zipcode RNA, which is required for proper RNA localization to cisternal-ER. RBP-P protein expression coincides with that of glutelin during seed maturation and is localized to both the nucleus and cytosol. RNA-immunoprecipitation and subsequent RT-PCR analysis further demonstrated that RBP-P interacts with glutelin RNAs. In vitro RNA–protein UV-crosslinking assays showed that recombinant RBP-P binds strongly to glutelin mRNA, and in particular, 3′ UTR and zipcode RNA. RBP-P also exhibited strong binding activity to a glutelin intron sequence, suggesting that RBP-P might participate in mRNA splicing. Overall, these results support a multifunctional role for RBP-P in glutelin mRNA metabolism, perhaps in nuclear pre-mRNA splicing and cytosolic localization to the cisternal-ER.     

Ribosomal Protein S14 of Saccharomyces cerevisiae Regulates Its Expression by Binding to RPS14B Pre-mRNA and to 18S rRNA

Production of ribosomal protein S14 in Saccharomyces cerevisiae is coordinated with the rate of ribosome assembly by a feedback mechanism that represses expression of RPS14B. Three-hybrid assays in vivo and filter binding assays in vitro demonstrate that rpS14 directly binds to an RNA stem-loop structure in RPS14B pre-mRNA that is necessary for RPS14B regulation. Moreover, rpS14 binds to a conserved helix in 18S rRNA with approximately five- to sixfold-greater affinity. These results support the model that RPS14B regulation is mediated by direct binding of rpS14 either to its pre-mRNA or to rRNA. Investigation of these interactions with the three-hybrid system reveals two regions of rpS14 that are involved in RNA recognition. D52G and E55G mutations in rpS14 alter the specificity of rpS14 for RNA, as indicated by increased affinity for RPS14B RNA but reduced affinity for the rRNA target. Deletion of the C terminus of rpS14, where multiple antibiotic resistance mutations map, prevents binding of rpS14 to RNA and production of functional 40S subunits. The emetine-resistant protein, rpS14-Em^RR, which contains two mutations near the C terminus of rpS14, does not bind either RNA target in the three-hybrid or in vitro assays. This is the first direct demonstration that an antibiotic resistance mutation alters binding of an r protein to rRNA and is consistent with the hypothesis that antibiotic resistance mutations can result from local alterations in rRNA structure.     

Evidence for a direct interaction of Rev protein with nuclear envelop mRNA-translocation system.

The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV-1) with the nucleocytoplasmic mRNA-transport system was investigated. In gel-shift assay, the recombinant Rev protein used in this study selectively bound to the Rev-responsive element (RRE) region of HIV-1 env-specific RNA. Nitrocellulose-filter-binding studies and Northern/Western-blotting experiments revealed an association constant of approximately 1 x 10(10) M-1. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)-binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)-rich mRNA. Binding of 125I-Rev to a 110-kDa nuclear-envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)-rich RNA, such as actin RNA [but not efflux of poly(A)-free RNA] from isolated nuclei and the nuclear-envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev-responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear-envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down-regulates the NTPase-driven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE-free RNA, ATP-dependent efflux of RRE-containing RNA from resealed nuclear-envelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE-containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear-envelope pore complex.     

Structure of the Y14-Magoh core of the exon junction complex

BACKGROUND: Splicing of pre-mRNA in eukaryotes imprints the resulting mRNA with a specific multiprotein complex, the exon-exon junction complex (EJC), at the sites of intron removal. The proteins of the EJC, Y14, Magoh, Aly/REF, RNPS1, Srm160, and Upf3, play critical roles in postsplicing processing, including nuclear export and cytoplasmic localization of the mRNA, and the nonsense-mediated mRNA decay (NMD) surveillance process. Y14 and Magoh are of particular interest because they remain associated with the mRNA in the same position after its export to the cytoplasm and require translation of the mRNA for removal. This tenacious, persistent, splicing-dependent, yet RNA sequence-independent, association suggests an important signaling function and must require distinct structural features for these proteins. RESULTS: We describe the high-resolution structure and biochemical properties of the highly conserved human Y14 and Magoh proteins. Magoh has an unusual structure comprised of an extremely flat, six-stranded anti-parallel beta sheet packed against two helices. Surprisingly, Magoh binds with high affinity to the RNP motif RNA binding domain (RBD) of Y14 and completely masks its RNA binding surface. CONCLUSIONS: The structure and properties of the Y14-Magoh complex suggest how the pre-mRNA splicing machinery might control the formation of a stable EJC-mRNA complex at splice junctions.     

Interactions of ribosomal protein S1 with DsrA and rpoS mRNA

Ribosomal protein S1 is shown to interact with the non-coding RNA DsrA and with rpoS mRNA. DsrA is a non-coding RNA that is important in controlling expression of the rpoS gene product in Escherichia coli. Photochemical crosslinking, quadrupole-time of flight tandem mass spectrometry, and peptide sequencing have identified an interaction between DsrA and S1 in the 30S ribosomal subunit. Purified S1 binds both DsrA (K(obs) approximately 6 x 10(6) M(-1)) and rpoS mRNA (K(obs) approximately 3 x 10(7) M(-1)). Ribonuclease probing experiments indicate that S1 binding has a weak but detectable effect on the secondary structure of DsrA or rpoS mRNA.     

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.