Molecular chaperones and quality control in noncoding RNA biogenesis.

Although noncoding RNAs have critical roles in all cells, both the mechanisms by which these RNAs fold into functional structures and the quality control pathways that monitor correct folding are only beginning to be elucidated. Here, we discuss several proteins that likely function as molecular chaperones for noncoding RNAs and review the existing knowledge on noncoding RNA quality control. One protein, the La protein, binds many nascent noncoding RNAs in eukaryotes and is required for efficient folding of certain pre-tRNAs. In prokaryotes, the Sm-like protein Hfq is required for the function of many noncoding RNAs. Recent work in bacteria and yeast has revealed the existence of quality control systems involving polyadenylation of unstable noncoding RNAs followed by exonucleolytic degradation. In addition, the Ro protein, which is present in many animal cells and also certain bacteria, binds misfolded noncoding RNAs and is proposed to function in RNA quality control.

Phosphorylation of the Saccharomyces cerevisiae La protein does not appear to be required for its functions in tRNA maturation and nascent RNA stabilization.

An abundant nuclear phosphoprotein, the La autoantigen, is the first protein to bind all newly synthesized RNA polymerase III transcripts. Binding by the La protein to the 3' ends of these RNAs stabilizes the nascent transcripts from exonucleolytic degradation. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the La protein is required for the normal pathway of tRNA maturation. Experiments in which the human protein was expressed in S. pombe have suggested that phosphorylation of the La protein regulates tRNA maturation. To dissect the role of phosphorylation in La protein function, we used mass spectrometry to identify three sites of serine phosphorylation in the S. cerevisiae La protein Lhp1p. Mutant versions of Lhp1p, in which each of the serines was mutated to alanine, were expressed in yeast cells lacking Lhp1p. Using two-dimensional gel electrophoresis, we determined that we had identified and mutated all major sites of phosphorylation in Lhp1p. Lhp1p lacking all three phosphorylation sites was functional in several yeast strains that require Lhp1p for growth. Northern blotting revealed no effects of Lhp1p phosphorylation status on either pre-tRNA maturation or stabilization of nascent RNAs. Both wild-type and mutant Lhp1 proteins localized to both nucleoplasm and nucleoli, demonstrating that phosphorylation does not affect subcellular location. Thus, although La proteins from yeast to humans are phosphoproteins, phosphorylation does not appear to be required for any of the identified functions of the S. cerevisiae protein.

Multiple functional interactions between components of the Lsm2-Lsm8 complex, U6 snRNA, and the yeast La protein.

The U6 small nuclear ribonucleoprotein is a critical component of the eukaryotic spliceosome. The first protein that binds the U6 snRNA is the La protein, an abundant phosphoprotein that binds the 3' end of many nascent small RNAs. A complex of seven Sm-like proteins, Lsm2-Lsm8, also binds the 3' end of U6 snRNA. A mutation within the Sm motif of Lsm8p causes Saccharomyces cerevisiae cells to require the La protein Lhp1p to stabilize nascent U6 snRNA. Here we describe functional interactions between Lhp1p, the Lsm proteins, and U6 snRNA. LSM2 and LSM4, but not other LSM genes, act as allele-specific, low-copy suppressors of mutations in Lsm8p. Overexpression of LSM2 in the lsm8 mutant strain increases the levels of both Lsm8p and U6 snRNPs. In the presence of extra U6 snRNA genes, LSM8 becomes dispensable for growth, suggesting that the only essential function of LSM8 is in U6 RNA biogenesis or function. Furthermore, deletions of LSM5, LSM6, or LSM7 cause LHP1 to become required for growth. Our experiments are consistent with a model in which Lsm2p and Lsm4p contact Lsm8p in the Lsm2-Lsm8 ring and suggest that Lhp1p acts redundantly with the entire Lsm2-Lsm8 complex to stabilize nascent U6 snRNA.

Sm-like proteins wRING the neck of mRNA.

Ring-shaped structures containing seven Sm or Sm-like proteins are stable components of several small nuclear ribonucleoprotein particles that function in pre-mRNA splicing. Recent reports describe a role for a distinct complex of seven Sm-like proteins in a very different process: mRNA degradation.

U snRNP assembly in yeast involves the La protein.

In all eukaryotic nuclei, the La autoantigen binds nascent RNA polymerase III transcripts, stabilizing these RNAs against exonucleases. Here we report that the La protein also functions in the assembly of certain RNA polymerase II-transcribed RNAs into RNPs. A mutation in a core protein of the spliceosomal snRNPs, Smd1p, causes yeast cells to require the La protein Lhp1p for growth at low temperatures. Precursors to U1, U2, U4 and U5 RNAs are bound by Lhp1p in both wild-type and mutant cells. At the permissive temperature, smd1-1 cells contain higher levels of stable U1 and U5 snRNPs when Lhp1p is present. At low temperatures, Lhp1p becomes essential for the accumulation of U4/U6 snRNPs and for cell viability. When U4 RNA is added to extracts, the pre-U4 RNA, but not the mature RNA, is bound by Smd1p. These results suggest that, by stabilizing a 3'-extended form of U4 RNA, Lhp1p facilitates efficient Sm protein binding, thus assisting formation of the U4/U6 snRNP.

Ro ribonucleoproteins contribute to the resistance of Deinococcus radiodurans to ultraviolet irradiation.

The genome of the radiation-resistant eubacterium Deinococcus radiodurans contains an ortholog of an RNA-binding protein known as the Ro 60-kD autoantigen. This protein, which was previously identified only in higher eukaryotes, is normally bound to small RNAs known as Y RNAs. We show that the Ro protein ortholog Rsr contributes to the resistance of D. radiodurans to UV irradiation. Rsr binds several small RNAs, encoded upstream of rsr, that accumulate following UV irradiation. One of these RNAs resembles a Y RNA. These results suggest that Ro RNPs could similarly contribute to the recovery of higher cells following UV irradiation.

Import of proteins into the trypanosome nucleus and their distribution at karyokinesis.

In all eukaryotic organisms proteins are targeted to the nucleus via a receptor-mediated mechanism that requires a specific nuclear localization sequence (NLS) in the protein. Little is known about this process in trypanosomatid protozoa that are considered amongst the earliest divergent eukaryotes. We have used the green fluorescent protein (gfp) and beta-galactosidase reporters to identify the NLS of two trypanosomal proteins, namely the Trypanosoma brucei La protein homologue and histone H2B of T. cruzi. A monopartite NLS was demonstrated at the C terminus of the La protein, whereas a bipartite NLS was identified within the first 40 amino acids of histone H2B. Treatment of live trypanosomes with poisons of ATP synthesis resulted in exit of the La NLS-gfp fusion from the nucleus. Interestingly, this fusion protein accumulated at several discrete sites in the cytoplasm, rather than equilibrating between the nucleus and the cytoplasm. When ATP levels returned to normal, the protein reentered the nucleus, demonstrating that the process was energy dependent. Finally, using fusion proteins that localize to the nucleoplasm or the nucleolus, we identified a subpopulation of mitotic cells in which the chromosomes have segregated but the daughter nuclei remain connected by a thin thread-like structure. We propose that cells containing this structure represent a late stage in nuclear division that can be placed after chromosome segregation, but before completion of karyokinesis.

Two yeast La motif-containing proteins are RNA-binding proteins that associate with polyribosomes.

We have characterized two Saccharomyces cerevisiae proteins, Sro9p and Slf1p, which contain a highly conserved motif found in all known La proteins. Originally described as an autoantigen in patients with rheumatic disease, the La protein binds to newly synthesized RNA polymerase III transcripts. In yeast, the La protein homologue Lhp1p is required for the normal pathway of tRNA maturation and also stabilizes newly synthesized U6 RNA. We show that deletions in both SRO9 and SLF1 are not synthetically lethal with a deletion in LHP1, indicating that the three proteins do not function in a single essential process. Indirect immunofluorescence microscopy reveals that although Lhp1p is primarily localized to the nucleus, Sro9p is cytoplasmic. We demonstrate that Sro9p and Slf1p are RNA-binding proteins that associate preferentially with translating ribosomes. Consistent with a role in translation, strains lacking either Sro9p or Slf1p are less sensitive than wild-type strains to certain protein synthesis inhibitors. Thus, Sro9p and Slf1p define a new and possibly evolutionarily conserved class of La motif-containing proteins that may function in the cytoplasm to modulate mRNA translation.

Saccharomyces cerevisiae telomerase is an Sm small nuclear ribonucleoprotein particle.

Activation of the chromosome end-replicating enzyme telomerase can greatly extend the lifespan of normal human cells and is associated with most human cancers. In all eukaryotes examined, telomerase has an RNA subunit, a conserved reverse transcriptase subunit and additional proteins, but little is known about the assembly of these components. Here we show that the Saccharomyces cerevisiae telomerase RNA has a 5'-2,2,7-trimethylguanosine (TMG) cap and a binding site for the Sm proteins, both hallmarks of small nuclear ribonucleoprotein particles (snRNPs) that are involved in nuclear messenger RNA splicing. Immunoprecipitation of telomerase from yeast extracts shows that Sm proteins are assembled on the RNA and that most or all of the telomerase activity is associated with the Sm-containing complex. These data support a model in which telomerase RNA is transcribed by RNA polymerase II and 7-methylguanosine-capped, binds the seven Sm proteins, becomes TMG-capped and picks up the other protein subunits. We conclude that the functions of snRNPs assembled by this pathway are not restricted to RNA processing, but also include chromosome telomere replication.

A role for the yeast La protein in U6 snRNP assembly: evidence that the La protein is a molecular chaperone for RNA polymerase III transcripts.

The first protein that binds to all newly synthesized RNA polymerase III transcripts is a highly conserved phosphoprotein known as the La autoantigen. Although binding by the yeast La protein Lhp1p to pre-tRNAs is required for the normal pathway of tRNA maturation, the role of the La protein in the biogenesis of other polymerase III transcripts has been unclear. We identified a mutation in a novel component of the U6 snRNP that causes yeast cells to require Lhp1p for growth. This protein, Lsm8p, is a member of a family of proteins, known as Sm-like proteins, that shares two conserved motifs with the core Sm proteins of the U1, U2, U4 and U5 snRNPs. The lsm8-1 cells have drastically reduced levels of the mature U6 snRNP, consistent with a defect in U6 snRNP assembly. In these cells, Lhp1p stabilizes newly synthesized U6 RNA, thus facilitating assembly of the RNA into the U6 snRNP. These results provide evidence that Lhp1p is a molecular chaperone for polymerase III-transcribed RNAs and implicate Lsm8p as a key component in the very early steps of U6 snRNP assembly.

Binding of the 60-kDa Ro autoantigen to Y RNAs: evidence for recognition in the major groove of a conserved helix.

The 60-kDa Ro autoantigen is normally complexed with small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the Ro protein is also complexed with a large class of variant 5S rRNA precursors that are folded incorrectly. Using purified baculovirus-expressed protein, we show that the 60-kDa Ro protein binds directly to both Y RNAs and misfolded 5S rRNA precursors. To understand how the protein recognizes these two distinct classes of RNAs, we investigated the features of Y RNA sequence and structure that are necessary for protein recognition. We identified a truncated Y RNA that is stably bound by the 60-kDa Ro protein. Within this 39-nt RNA is a conserved helix that is proposed to be the binding site for the Ro protein. Mutagenesis of this minimal Y RNA revealed that binding by the 60-kDa Ro protein requires specific base pairs within the conserved helix, a singly bulged nucleotide that disrupts the helix, and a three-nucleotide bulge on the opposing strand. Chemical probing experiments using diethyl pyrocarbonate demonstrated that, in the presence of the two bulges, the major groove of the conserved helix is accessible to protein side chains. These data are consistent with a model in which the Ro protein recognizes specific base pairs in the conserved helix by binding in the major groove of the RNA. Furthermore, experiments in which dimethyl sulfate was used to probe a naked and protein-bound Y RNA revealed that a structural alteration occurs in the RNA upon Ro protein binding.

The La protein in Schizosaccharomyces pombe: a conserved yet dispensable phosphoprotein that functions in tRNA maturation.

Most RNA polymerase III transcripts are bound immediately after synthesis by an abundant nuclear phosphoprotein known as the La autoantigen. Experiments performed in the budding yeast Saccharomyces cerevisiae have revealed that binding of the La protein to tRNA precursors is required for the endonucleolytic maturation of the 3' terminus of many tRNAs. In the absence of this protein, the 3' ends of these tRNAs are trimmed by exonucleases (Yoo CJ, Wolin SL, 1997, Cell 89:393-402). Here we report the characterization of the La protein in the fission yeast Schizosaccharomyces pombe. As was described for budding yeast, S. pombe cells lacking the La protein are viable and exhibit alterations in the pathway of pre-tRNA maturation. Introduction of either the human, S. cerevisiae, or S. pombe La protein into these cells restores the detected pattern of tRNA processing intermediates to that of wild-type cells. By performing immunoprecipitations from cells that were metabolically labeled with 32P-orthophosphate, we demonstrate that the S. pombe and S. cerevisiae La proteins, like the human La protein, are phosphorylated in vivo. Thus, although the La protein is dispensable for growth in these yeasts, both the structure of the protein and its function in pre-tRNA maturation have been highly conserved throughout evolution.

The yeast La protein is required for the 3' endonucleolytic cleavage that matures tRNA precursors.

Although the La autoantigen binds to the 3' ends of all nascent polymerase III transcripts, its function in vivo has long been unclear. Although S. cerevisiae cells lacking the La protein homolog Lhp1p are viable, cells containing a mutation that disrupts the anticodon stem of tRNA(Ser)CGA require Lhp1p for growth. We demonstrate that for the wild-type pre-tRNA(Ser)CGA and other pre-tRNAs, Lhp1p is required for the normal endonucleolytic removal of the 3' trailer sequence. In cells lacking Lhp1p, the 3' trailer is removed by exonuclease(s). Although maturation of the mutant pre-tRNA(Ser)CGA requires Lhp1p, introduction of a second mutation that restores base pairing eliminates the requirement. We propose that binding by Lhp1p stabilizes pre-tRNAs in conformations that allow the 3' endonucleolytic cleavage to occur.

A misfolded form of 5S rRNA is complexed with the Ro and La autoantigens.

In both vertebrate and invertebrate cells, the 60-kDa Ro autoantigen is bound to small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the 60-kDa Ro protein is also complexed with a class of 5S rRNA precursors that contain internal mutations. Because these 5S rRNA precursors are processed inefficiently and degraded eventually, the Ro protein may function in a quality control pathway for 5S rRNA biosynthesis. We have investigated the sequence and secondary structure determinants in the mutant 5S rRNAs that confer binding by the 60-kDa Ro protein. The mutant 5S rRNAs fold to form an alternative helix that is required for recognition by the 60-kDa Ro protein. Mutations that disrupt the alternative helix eliminate Ro protein binding, whereas compensatory changes that restore the helix are bound efficiently by the Ro protein. When the structure of the mutant RNA was probed using dimethylsulfate and oligonucleotide-directed RNase H cleavage, the results were consistent with the formation of the alternative structure. The La protein, which is also complexed with the mutant 5S rRNA precursors, protects similar sequences from nuclease digestion as does the 60-kDa Ro protein. Thus, the binding sites for these two proteins are either nearby on the RNA, or the two proteins may be complexed through protein-protein interactions. When the human Ro protein is expressed in the yeast Saccharomyces cerevisiae, the protein binds wild-type 5S rRNA precursors, suggesting that a population of wild-type precursors also folds into the alternative structure.

A perinucleolar compartment contains several RNA polymerase III transcripts as well as the polypyrimidine tract-binding protein, hnRNP I.

We have investigated the subcellular organization of the four human Y RNAs. These RNAs, which are transcribed by RNA polymerase III, are usually found complexed with the Ro autoantigen, a 60-kD protein. We designed 2'-OMe oligoribonucleotides that were complementary to accessible single-stranded regions of Y RNAs within Ro RNPs and used them in fluorescence in situ hybridization. Although all four Y RNAs were primarily cytoplasmic, oligonucleotides directed against three of the RNAs hybridized to discrete structures near the nucleolar rim. We have termed these structures "perinucleolar compartments" (PNCs). Double labeling experiments with appropriate antisera revealed that PNCs are distinct from coiled bodies and fibrillar centers. Co-hybridization with a genomic DNA clone spanning the human Y1 and Y3 genes showed that PNCs are not stably associated with the transcription site for these Y RNAs. Although 5S rDNA was often located near the nucleolar periphery, PNCs are not associated with 5S gene loci. Two additional pol III transcripts, the RNA components of RNase P and RNase MRP, did colocalize within PNCs. Most interestingly, the polypyrimidine tract-binding protein hnRNP I/PTB was also concentrated in this compartment. Possible roles for this novel nuclear subdomain in macromolecular assembly and/or nucleocytoplasmic shuttling of these five pol III transcripts, along with hnRNP I/PTB, are discussed.

Caenorhabditis elegans embryos contain only one major species of Ro RNP.

In virtually all vertebrate cells, Ro RNPs consist of the 60-kDa Ro autoantigen bound to one of several small cytoplasmic RNA molecules known as Y RNAs. Because the 60-kDa Ro autoantigen is also found complexed with defective precursors of 5S rRNA in Xenopus oocytes, we have proposed that this protein functions in a quality control, or discard pathway, for 5S RNA biosynthesis (O'Brien CA, Wolin SL, 1994, Genes & Dev 8:2891-2903). The role of the Y RNAs in this pathway is unknown. To begin a genetic analysis of Ro RNP function, we have characterized these particles in the nematode Caenorhabditis elegans. The C. elegans Ro protein is 12 kDa larger than the vertebrate protein; the larger size is due in part to an N-terminal extension and to two insertions in the RNA recognition motif. In contrast to all previously described vertebrate species, the Ro protein appears bound to a single Y RNA in C. elegans. Similar to vertebrate Y RNAs, the C. elegans Y RNA can be folded to form a pyrimidine-rich internal loop and a long stem in which the 5' and 3' ends are base paired. Within the stem is a conserved bulged helix that is proposed to be the binding site of the Ro protein. Interestingly, although the human protein can bind the nematode Y RNA, the C. elegans protein does not bind human Y RNAs. This is the first description of Ro RNPs in an invertebrate species.

A possible role for the 60-kD Ro autoantigen in a discard pathway for defective 5S rRNA precursors.

The Ro autoantigen is a 60-kD protein that is usually found in small cytoplasmic RNA-protein complexes known as Ro RNPs. Although the Ro RNPs are abundant and conserved components of a variety of vertebrate and invertebrate cells, their function is unknown. We have discovered that the Ro protein is also found complexed with certain variant 5S rRNAs in Xenopus oocytes. These RNAs contain one or more point mutations compared with the major oocyte 5S rRNA sequence as well as additional nucleotides at the 3' end. We demonstrate that the Ro protein binds specifically mutant 5S rRNAs containing 3' terminal extensions. These mutant RNAs are processed inefficiently to mature 5S rRNA and most eventually are degraded. The observation that the Ro autoantigen specifically associates with defective 5S rRNA precursors suggests that this protein may function as part of a novel quality control or discard pathway for 5S rRNA production.

La proteins from Drosophila melanogaster and Saccharomyces cerevisiae: a yeast homolog of the La autoantigen is dispensable for growth.

The human autoantigen La is a 50-kDa protein which binds to the 3' termini of virtually all nascent polymerase III transcripts. Experiments with mammalian transcription extracts have led to the proposal that the La protein is required for multiple rounds of transcription by RNA polymerase III (E. Gottlieb and J. A. Steitz, EMBO J. 8:851-861, 1989; R. J. Maraia, D. J. Kenan, and J. D. Keene, Mol. Cell. Biol. 14:2147-2158, 1994). Although La protein homologs have been identified in a variety of vertebrate species, the protein has not been identified in invertebrates. In order to begin a genetic analysis of La protein function, we have characterized homologs of the La protein in the fruit fly Drosophila melanogaster and the yeast Saccharomyces cerevisiae. We show that both the Drosophila and yeast La proteins are bound to precursors of polymerase III RNAs in vivo. The Drosophila and yeast proteins resemble the human La protein in their biochemical properties, as both proteins can be partially purified from cells by a procedure previously devised to purify the human protein. Similarly to vertebrate La proteins, the Drosophila and yeast homologs preferentially bind RNAs that terminate with a 3' hydroxyl. Despite the fact that the La protein is conserved between humans and Saccharomyces cerevisiae, yeast cells containing a null allele of the gene encoding the La protein are viable, suggesting that another protein(s) plays a functionally redundant role.