An improved definition of the RNA-binding specificity of SECIS-binding protein 2, an essential component of the selenocysteine incorporation machinery.

By binding to SECIS elements located in the 3'-UTR of selenoprotein mRNAs, the protein SBP2 plays a key role in the assembly of the selenocysteine incorporation machinery. SBP2 contains an L7Ae/L30 RNA-binding domain similar to that of protein 15.5K/Snu13p, which binds K-turn motifs with a 3-nt bulge loop closed by a tandem of G.A and A.G pairs. Here, by SELEX experiments, we demonstrate the capacity of SBP2 to bind such K-turn motifs with a protruding U residue. However, we show that conversion of the bulge loop into an internal loop reinforces SBP2 affinity and to a greater extent RNP stability. Opposite variations were found for Snu13p. Accordingly, footprinting assays revealed strong contacts of SBP2 with helices I and II and the 5'-strand of the internal loop, as opposed to the loose interaction of Snu13p. Our data also identifies new determinants for SBP2 binding which are located in helix II. Among the L7Ae/L30 family members, these determinants are unique to SBP2. Finally, in accordance with functional data on SECIS elements, the identity of residues at positions 2 and 3 in the loop influences SBP2 affinity. Altogether, the data provide a very precise definition of the SBP2 RNA specificity.

Selenoprotein synthesis: UGA does not end the story.

It is well established that the beneficial effects of the trace element selenium are mediated by its major biological product, the amino acid selenocysteine, present in the active site of selenoproteins. These fulfill different functions, as varied as oxidation-reduction of metabolites in bacteria, reduction of reactive oxygen species, control of the redox status of the cell or thyroid hormone maturation. This review will focus on the singularities of the selenocysteine biosynthesis pathway and its unique incorporation mechanism into eukaryal selenoproteins. Selenocysteine biosynthesis from serine is achieved on tRNA(Sec) and requires four proteins. As this amino acid is encoded by an in-frame UGA codon, otherwise signaling termination of translation, ribosomes must be told not to stop at this position in the mRNA. Several molecular partners acting in cis or in trans have been identified, but their knowledge has not enabled yet to firmly establish the molecular events underlying this mechanism. Data suggest that other, so far uncharacterized factors might exist. In this survey, we attempted to compile all the data available in the literature and to describe the latest developments in the field.

Three novel components of the human exosome.

The yeast exosome is a complex of 3' --> 5' exoribonucleases. Sequence analysis identified putative human homologues for exosome components, although several were found only as expressed sequence tags. Here we report the cloning of full-length cDNAs, which encode putative human homologues of the Rrp40p, Rrp41p, and Rrp46p components of the exosome. Recombinant proteins were expressed and used to raise rabbit antisera. In Western blotting experiments, these decorated HeLa cell proteins of the predicted sizes. All three human proteins were enriched in the HeLa cells nucleus and nucleolus, but were also clearly detected in the cytoplasm. Size exclusion chromatography revealed that hRrp40p, hRrp41p, and hRrp46p were present in a large complex. This cofractionated with the human homologues of other exosome components, hRrp4p and PM/Scl-100. Anti-PM/Scl-positive patient sera coimmunoprecipitated hRrp40p, hRrp41p, and hRrp46p demonstrating their physical association. The immunoprecipitated complex exhibited 3' --> 5' exoribonuclease activity in vitro. hRrp41p was expressed in yeast and shown to suppress the lethality of genetic depletion of yeast Rrp41p. We conclude that hRrp40p, hRrp41p, and hRrp46p represent novel components of the human exosome complex.

Precursors to the U3 small nucleolar RNA lack small nucleolar RNP proteins but are stabilized by La binding.

Almost all small eukaryotic RNAs are processed from transiently stabilized 3'-extended forms. A key question is how and why such intermediates are stabilized and how they can then be processed to the mature RNA. Here we report that yeast U3 is also processed from a 3'-extended precursor. The major 3'-extended forms of U3 (U3-3'I and -II) lack the cap trimethylation present in mature U3 and are not associated with small nucleolar RNP (snoRNP) proteins that bind mature U3, i.e., Nop1p, Nop56p, and Nop58p. Depletion of Nop58p leads to the loss of mature U3 but increases the level of U3-3'I and -II, indicating a requirement for the snoRNP proteins for final maturation. Pre-U3 is cleaved by the endonuclease Rnt1p, but U3-3'I and -II do not extend to the Rnt1p cleavage sites. Rather, they terminate at poly(U) tracts, suggesting that they might be bound by Lhp1p (the yeast homologue of La). Immunoprecipitation of Lhp1p fused to Staphylococcus aureus protein A resulted in coprecipitation of both U3-3'I and -II. Deletion of LHP1, which is nonessential, led to the loss of U3-3'I and -II. We conclude that pre-U3 is cleaved by Rnt1p, followed by exonuclease digestion to U3-3'I and -II. These species are stabilized against continued degradation by binding of Lhp1p. Displacement of Lhp1p by binding of the snoRNP proteins allows final maturation, which involves the exosome complex of 3'-->5' exonucleases.

Degradation of ribosomal RNA precursors by the exosome.

The yeast exosome is a complex of 3'-->5' exonucleases involved in RNA processing and degradation. All 11 known components of the exosome are required during 3' end processing of the 5.8S rRNA. Here we report that depletion of each of the individual components inhibits the early pre-rRNA cleavages at sites A(0), A(1), A(2)and A(3), reducing the levels of the 32S, 20S, 27SA(2)and 27SA(3)pre-rRNAs. The levels of the 27SB pre-rRNAs were also reduced. Consequently, both the 18S and 25S rRNAs were depleted. Since none of these processing steps involves 3'-->5' exonuclease activities, the requirement for the exosome is probably indirect. Correct assembly of trans -acting factors with the pre-ribosomes may be monitored by a quality control system that inhibits pre-rRNA processing. The exosome itself degrades aberrant pre-rRNAs that arise from such inhibition. Exosome mutants stabilize truncated versions of the 23S, 21S and A(2)-C(2)RNAs, none of which are observed in wild-type cells. The putative helicase Dob1p, which functions as a cofactor for the exosome in pre-rRNA processing, also functions in these pre-rRNA degradation activities.

Functions of the exosome in rRNA, snoRNA and snRNA synthesis.

The yeast nuclear exosome contains multiple 3'-->5' exoribonucleases, raising the question of why so many activities are present in the complex. All components are required during the 3' processing of the 5.8S rRNA, together with the putative RNA helicase Dob1p/Mtr4p. During this processing three distinct steps can be resolved, and hand-over between different exonucleases appears to occur at least twice. 3' processing of snoRNAs (small nucleolar RNAs) that are excised from polycistronic precursors or from mRNA introns is also a multi-step process that involves the exosome, with final trimming specifically dependent on the Rrp6p component. The spliceosomal U4 snRNA (small nuclear RNA) is synthesized from a 3' extended precursor that is cleaved by Rnt1p at sites 135 and 169 nt downstream of the mature 3' end. This cleavage is followed by 3'-->5' processing of the pre-snRNA involving the exosome complex and Dob1p. The exosome, together with Rnt1p, also participates in the 3' processing of the U1 and U5 snRNAs. We conclude that the exosome is involved in the processing of many RNA substrates and that different components can have distinct functions.

The yeast exosome and human PM-Scl are related complexes of 3' --> 5' exonucleases.

We previously identified a complex of 3' --> 5' exoribonucleases, designated the exosome, that is expected to play a major role in diverse RNA processing and degradation pathways. Further biochemical and genetic analyses have revealed six novel components of the complex. Therefore, the complex contains 11 components, 10 of which are predicted to be 3' --> 5' exoribonucleases on the basis of sequence homology. Human homologs were identified for 9 of the 11 yeast exosome components, three of which complement mutations in the respective yeast genes. Two of the newly identified exosome components are homologous to known components of the PM-Scl particle, a multisubunit complex recognized by autoimmune sera of patients suffering from polymyositis-scleroderma overlap syndrome. We demonstrate that the homolog of the Rrp4p exosome subunit is also a component of the PM-Scl complex, thereby providing compelling evidence that the yeast exosome and human PM-Scl complexes are functionally equivalent. The two complexes are similar in size, and biochemical fractionation and indirect immunofluorescence experiments show that, in both yeast and humans, nuclear and cytoplasmic forms of the complex exist that differ only by the presence of the Rrp6p/PM-Scl100 subunit exclusively in the nuclear complex.

The role of the 3' external transcribed spacer in yeast pre-rRNA processing.

We have undertaken a deletion analysis of the 3' external transcribed spacer (3' ETS) in the pre-rRNA of Saccharomyces cerevisiae. A stem loop structure immediately 3' to the 25 S rRNA region is necessary and sufficient for processing of the 3' ETS. This is believed to be by cotranscriptional cleavage by Rnt1p, the yeast homologue of RNase III. In addition, this stem-loop is required for cleavage of site A3 by RNase MRP and for processing at site B1L, in the 3' region of ITS1. Processing at an upstream site in ITS1, site A2, and at sites in the 5' external transcribed spacer are not affected, even by complete deletion of the 3' ETS. We conclude that processing in the 3' ETS and in ITS1 is coupled. This would constitute a quality control that prevents synthesis of the 5. 8 S rRNA and 5' end maturation of the 25 S rRNA in transcripts which are incomplete due to premature transcription termination.

Accurate processing of a eukaryotic precursor ribosomal RNA by ribonuclease MRP in vitro.

Very few of the enzymes required for eukaryotic precursor ribosomal RNA (pre-rRNA) processing have been identified. Ribonuclease (RNase) MRP was characterized as a nuclease that cleaves mitochondrial replication primers, but it is predominantly nucleolar. Previous genetic evidence revealed that this ribonucleoprotein is required, directly or indirectly, for cleavage of the yeast pre-rRNA in vivo at site A3. Here, an in vitro processing system that accurately reproduces this cleavage is described. Biochemical purification and the use of extracts depleted of the MRP RNA demonstrate that endonucleolytic cleavage of the pre-rRNA is directly mediated by RNase MRP. This establishes a role for RNase MRP in the nucleolus.

Recognition of cleavage site A(2) in the yeast pre-rRNA.

Processing of the yeast pre-rRNA at site A(2) internal transcribed spacer 1(ITS1) has been shown to require several small nucleolar ribonucleoprotein particles (snoRNPs) as trans-acting factors. Here we report a detailed mutational analysis of the cid-acting signals required to specify the site of A(2) lie in the 3'-flanking sequence; deletion or substitution of nucleotides in this region strongly inhibits processing, and residual cleavage is inaccurate at the nucleotide level. In contrast, the deletion of the 5'- flanking nucleotides has no detectable effect on processing. An evolutionarily conserved sequence, ACAC, is located at the site of cleavage. Substitution of the 3' AC leads to heterogeneous cleavage, with activation of cleavage at an upstream ACAC sequence, In all mutants that retain an ACAC element, a site of cleavage is detected immediately 5' to this sequence, showing that this element is recognized. An ACAC sequence is, however, not essential for accurate cleavage of site A(2). An additional signal is also present 3' to A(2), in a region that has the potential to form a stem-loop structure that is evolutionarily conserved, but of low stability. As has been found for site A(1) (the 5' end of the yeast 18S rRNA), the identification of the site of processing at A(2) relies on multiple recognition elements.

Processing of the yeast pre-rRNA at sites A(2) and A(3) is linked.

Cleavage of the yeast pre-rRNA at site A(2) in internal transcribed spacer 1 (ITS1) requires multiple snoRNP species, whereas cleavage at site A(3),located 72 nt 3' in ITS1, requires Rnase MRP. Analyses of mutations in the pre- rRNA have revealed an unexpected link between processing at A(2) and A(3). Small substitution mutations in the 3' flanking sequence at A(2) inhibit processing at site A(3), whereas a small deletion at A(3) has been shown to delay processing at site A(2). Moreover, the combination of mutations in cis at both A(2) and A(3) leads to the synthesis of pre-rRNA species with 5' ends within the mature 18S rRNA sequence, at sites between + 482 and + 496. The simultaneous interference with an snoRNP processing complex at site A(2) and an Rnase MPRP complex at site A(3) may activate a pre-rRNA breakdown pathway. The same aberantpre-rRNA species are observed in strains with mutations in the RNA component of Rnase MRP, consistent with interactions between the processing complexes. Furthermore, genetic depletion of the snoRNA, snR30, has been shown to affect the coupling between cleavage by Rnase MRP and subsequent exonuclease digestion.We conclude that an sno-RNP-dependent processing complex that is required for A(2) cleavage and that recognizes the 3' flanking sequence at A(2), interacts with the RNase MRP complex bound to the pre-rRNA around site A(3).

Role of conserved nucleotides in building the 16S rRNA binding site of E. coli ribosomal protein S8.

Ribosomal protein S8 specifically recognizes a helical and irregular region of 16S rRNA that is highly evolutionary constrained. Despite its restricted size, the precise conformation of this region remains a question of debate. Here, we used chemical probing to analyze the structural consequences of mutations in this RNA region. These data, combined with computer modelling and previously published data on protein binding were used to investigate the conformation of the RNA binding site. The experimental data confirm the model in which adenines A595, A640 and A642 bulge out in the deep groove. In addition to the already proposed non canonical U598-U641 interaction, the structure is stabilized by stacking interactions (between A595 and A640) and an array of hydrogen bonds involving bases and the sugar phosphate backbone. Mutations that alter the ability to form these interdependent interactions result in a local destabilization or reorganization. The specificity of recognition by protein S8 is provided by the irregular and distorted backbone and the two bulged adenines 640 and 642 in the deep groove. The third adenine (A595) is not a direct recognition site but must adopt a bulged position. The U598-U641 pair should not be directly in contact with the protein.

Minimal 16S rRNA binding site and role of conserved nucleotides in Escherichia coli ribosomal protein S8 recognition.

Escherichia coli ribosomal protein S8 was previously shown to bind a 16S rRNA fragment (nucleotides 584-756) with the same affinity as the complete 16S rRNA, and to shield an irregular helical region (region C) [Mougel, M., Eyermann, F., Westhof, E., Romby, P., Expert-Bezançon, Ebel, J. P., Ehresmann, B. & Ehresmann, C. (1987). J. Mol. Biol. 198, 91-107]. Region C was postulated to display characteristic features: three bulged adenines (A595, A640 and A642), a non-canonical U598-U641 pair surrounded by two G.C pairs. In order to delineate the minimal RNA binding site, deletions were introduced by site-directed mutagenesis and short RNA fragments were synthesized. Their ability to bind S8 was assayed by filter binding. Our results show that the RNA binding site can be restricted to a short helical stem (588-605/633-651) containing region C. The second part of the work focused on region C and on the role of conserved nucleotides as potential determinants of S8 recognition. Single and double mutations were introduced by site-directed mutagenesis in fragment 584-756, and their effect on S8 binding was measured. It was found that the three bulged positions are essential and that adenines are required at positions 640 and 642. U598 is also crucial and the highly conserved G597.C643 pair cannot be inverted. These conserved nucleotides are either directly involved in the recognition process as direct contacts or required to maintain a specific conformation. The strong evolutionary pressure and the small number of positive mutants stress the high stringency of the recognition process.

The secondary structure of the 5'-noncoding region of beet necrotic yellow vein virus RNA 3: evidence for a role in viral RNA replication.

Secondary structure-sensitive chemical and enzymatic probes have been used to produce a model for the folding of the first 312 residues of the long 5'-noncoding region of beet necrotic yellow vein virus RNA 3. The structure consists of two major domains, one of which includes long distance base-pairing interactions between two short sequence elements (Box I and Box II) situated between positions 237 and 292 and complementary elements (Box I' and II') near the 5'-terminus. Previous studies have shown that base pairing between these sequence elements (in either the plus-strand or minus-strand RNA) is important for RNA 3 accumulation during infection. RNA 3 transcripts were produced containing mutations which preferentially disrupted Box II-II' base pairing in either the plus- or minus-strand. In infection experiments, transcripts with mutations which disrupted the Box II-II' interaction in the plus-strand structure replicated less efficiently than mutants in which the Box II-II' interaction was disrupted in the minus-strand. These findings indicate that the complex 5'-proximal plus-strand structure to which the Box II-II' interaction contributes comprises at least part of the promoter for plus-strand RNA synthesis.