Biomedical subjects

Sandra L Wolin

Publications and source records attributed to Sandra L Wolin.

11 recordsLinked to original sources

PubMed2006

Structural and biochemical basis for misfolded RNA recognition by the Ro autoantigen.

The Ro autoantigen is ring-shaped, binds misfolded noncoding RNAs and is proposed to function in quality control. Here we determine how Ro interacts with misfolded RNAs. Binding of Ro to misfolded precursor (pre)-5S ribosomal RNA requires a single-stranded 3' end and helical elements. As mutating most sequences of the helices and tail results in modest decreases in binding, Ro may be able to associate with a range of RNAs. Ro binds several other RNAs that contain single-stranded tails. A crystal structure of Ro bound to a misfolded pre-5S rRNA fragment reveals that the tail inserts into the cavity, while a helix binds on the surface. Most contacts of Ro with the helix are to the backbone. Mutagenesis reveals that Ro has an extensive RNA-binding surface. We propose that Ro uses this surface to scavenge RNAs that fail to bind their specific RNA-binding proteins.

Animals↗

PubMed2006

The La protein functions redundantly with tRNA modification enzymes to ensure tRNA structural stability.

Although the La protein stabilizes nascent pre-tRNAs from nucleases, influences the pathway of pre-tRNA maturation, and assists correct folding of certain pre-tRNAs, it is dispensable for growth in both budding and fission yeast. Here we show that the Saccharomyces cerevisiae La shares functional redundancy with both tRNA modification enzymes and other proteins that contact tRNAs during their biogenesis. La is important for growth in the presence of mutations in either the arginyl tRNA synthetase or the tRNA modification enzyme Trm1p. In addition, two pseudouridine synthases, PUS3 and PUS4, are important for growth in strains carrying a mutation in tRNA(Arg)(CCG) and are essential when La is deleted in these strains. Depletion of Pus3p results in accumulation of the aminoacylated mutant tRNA(Arg)(CCG) in nuclei, while depletion of Pus4p results in decreased stability of the mutant tRNA. Interestingly, the degradation of mutant unstable forms of tRNA(Arg)(CCG) does not require the Trf4p poly(A) polymerase, suggesting that yeast cells possess multiple pathways for tRNA decay. These data demonstrate that La functions redundantly with both tRNA modifications and proteins that associate with tRNAs to achieve tRNA structural stability and efficient biogenesis.

Amino Acyl-tRNA Synthetases↗

PubMed2005

The Ro 60 kDa autoantigen comes into focus: interpreting epitope mapping experiments on the basis of structure.

A conserved RNA-binding protein, the Ro 60 kDa (Ro60) autoantigen, is a major target of autoantibodies in patients suffering from the rheumatic diseases Sjogren's syndrome, systemic lupus erythematosus, subacute cutaneous lupus erythematosus and neonatal lupus erythematosus. In both mice and certain bacteria, Ro60 is important for cell survival following ultraviolet irradiation. Although the function of Ro60 was mysterious for many years, recent experiments have demonstrated that this protein binds misfolded noncoding RNAs in vertebrate cells and likely functions in a pathway by which defective RNAs are recognized and targeted for degradation. Recent structural studies have revealed that Ro60 is shaped like a doughnut with an inner hole. Noncoding RNAs called Y RNAs bind on the outer surface of the ring, while the single-stranded ends of misfolded RNAs likely bind within the hole. Comparison of the Ro60 structure with the results of epitope-mapping studies reveals that many of the currently identified epitopes recognized by patient sera overlap regions of Ro60 that function in RNA binding. Moreover, in some patients with anti-Ro60 antibodies, the initial antigenic epitope corresponds to a loop involved in binding single-stranded RNA in the central cavity.

Animals↗

PubMed2005

Structural insights into RNA quality control: the Ro autoantigen binds misfolded RNAs via its central cavity.

The Ro 60 kDa autoantigen is a major target of the immune response in patients with systemic lupus erythematosus. In vertebrate cells, Ro binds misfolded small RNAs and likely functions in RNA quality control. In eukaryotes and bacteria, Ro also associates with small RNAs called Y RNAs. We present structures of unliganded Ro and Ro complexed with two RNAs at 1.95 and 2.2 A resolution, respectively. Ro consists of a von Willebrand factor A domain and a doughnut-shaped domain composed of HEAT repeats. In the complex, a fragment of Y RNA binds on the outer surface of the HEAT-repeat ring, and single-stranded RNA binds in the toroid hole. Mutagenesis supports a binding site for misfolded RNAs that encompasses both sites, with a single-stranded end inserted into the toroid cavity. Our experiments suggest that one role of Y RNAs may be to regulate access of other RNAs to Ro.

Amino Acid Motifs↗

PubMed2004

An Lsm2-Lsm7 complex in Saccharomyces cerevisiae associates with the small nucleolar RNA snR5.

Sm-like (Lsm) proteins function in a variety of RNA-processing events. In yeast, the Lsm2-Lsm8 complex binds and stabilizes the spliceosomal U6 snRNA, whereas the Lsm1-Lsm7 complex functions in mRNA decay. Here we report that a third Lsm complex, consisting of Lsm2-Lsm7 proteins, associates with snR5, a box H/ACA snoRNA that functions to guide site-specific pseudouridylation of rRNA. Experiments in which the binding of Lsm proteins to snR5 was reconstituted in vitro reveal that the 3' end of snR5 is critical for Lsm protein recognition. Glycerol gradient sedimentation and sequential immunoprecipitation experiments suggest that the Lsm protein-snR5 complex is partly distinct from the complex formed by snR5 RNA with the box H/ACA proteins Gar1p and Nhp2p. Consistent with a separate complex, Lsm proteins are not required for the function of snR5 in pseudouridylation of rRNA. We demonstrate that in addition to their known nuclear and cytoplasmic locations, Lsm proteins are present in nucleoli. Taken together with previous findings that a small fraction of pre-RNase P RNA associates with Lsm2-Lsm7, our experiments suggest that an Lsm2-Lsm7 protein complex resides in nucleoli, contributing to the biogenesis or function of specific snoRNAs.

Base Sequence↗

PubMed2004

Structure of the La motif: a winged helix domain mediates RNA binding via a conserved aromatic patch.

The La protein is a ubiquitous nuclear phosphoprotein that recognizes the 3' uridylates found in all newly synthesized RNA polymerase III transcripts. La binding stabilizes these transcripts from exonucleases and may also assist their folding. Here we present the first structural insights into how the La protein specifically interacts with its RNA substrates. The most conserved region of the La protein is the La motif, a domain also found in several other RNA-binding proteins. We have determined the structure of the La motif from the Trypanosoma brucei La protein to 1.6 A resolution (PDB code 1S29). The La motif adopts a winged helix-turn-helix architecture that has a highly conserved patch of mainly aromatic surface residues. Mutagenesis experiments support a critical role for this patch in RNA binding and show that it partly determines binding specificity for RNAs ending in 3' hydroxyl, a defining characteristic of the La protein. These findings reveal that the La motif is essential for high-affinity binding and also contributes to specificity.

Amino Acid Motifs↗

PubMed2004

The Ro 60 kDa autoantigen: insights into cellular function and role in autoimmunity.

An RNA-binding protein, the Ro 60 kDa autoantigen, is a major target of the immune response in patients suffering from two systemic rheumatic diseases, systemic lupus erythematosus and Sjogren's syndrome. In lupus patients, anti-Ro antibodies are associated with photosensitive skin lesions and with neonatal lupus, a syndrome in which mothers with anti-Ro antibodies give birth to children with photosensitive skin lesions and a cardiac conduction defect, third degree heart block. In vertebrate cells, the Ro protein binds small RNAs of unknown function known as Y RNAs. Although the cellular function of Ro has long been mysterious, recent studies have implicated Ro in two distinct processes: small RNA quality control and the enhancement of cell survival following exposure to ultraviolet irradiation. Most interestingly, mice lacking the Ro protein develop an autoimmune syndrome that shares some features with systemic lupus erythematosus in patients, suggesting that the normal function of Ro may be important for the prevention of this autoimmune disease. In this review, we summarize recent progress towards understanding the role of the Ro 60 kDa protein and discuss whether the cellular function of Ro could be related to certain manifestations of lupus in patients.

Animals↗

PubMed2003

The Ro autoantigen binds misfolded U2 small nuclear RNAs and assists mammalian cell survival after UV irradiation.

The Ro 60 kDa autoantigen, an RNA binding protein, is a major target of the immune response in patients with systemic lupus erythematosus. As mice lacking Ro develop a lupus-like syndrome, Ro may be important for preventing autoimmunity. However, the cellular function of Ro, which binds small cytoplasmic RNAs of unknown function called Y RNAs, has been enigmatic. Ro has been proposed to function in 5S rRNA quality control based on experiments in Xenopus laevis oocytes, and a Ro ortholog enhances survival of the eubacterium Deinococcus radiodurans after ultraviolet irradiation. To test the general importance of these two observations for Ro function, we investigated the role of Ro in mammalian cells. We report that, in mouse embryonic stem (ES) cells, Ro binds variant spliceosomal U2 snRNAs. Expression of mouse U2 snRNAs in Xenopus oocytes reveals that binding occurs in nuclei and appears to involve recognition of misfolded RNA. Moreover, mouse ES cells lacking Ro exhibit decreased survival after ultraviolet irradiation. In irradiated cells, both Ro and a Y RNA accumulate in nuclei. We propose that Ro plays a general role in small RNA quality control and that this function is important for cell survival after ultraviolet irradiation.

Animals↗

PubMed2003

A La protein requirement for efficient pre-tRNA folding.

The La protein protects the 3' ends of many nascent small RNAs from exonucleases. Here we report that La is required for efficient folding of certain pre-tRNAs. A mutation in pre-tRNA(Arg)(CCG) causes yeast cells to be cold-sensitive and to require the La protein Lhp1p for efficient growth. When the mutant cells are grown at low temperature, or when Lhp1p is depleted, mature tRNA(Arg)(CCG) is not efficiently aminoacylated. The mutation causes the anticodon stem of pre-tRNA(Arg)(CCG) to misfold into an alternative helix in vitro. Intragenic suppressor mutations that disrupt the misfolded helix or strengthen the correct helix alleviate the requirement for Lhp1p, providing evidence that the anticodon stem misfolds in vivo. Chemical and enzymatic footprinting experiments suggest a model in which Lhp1p stabilizes the correctly folded stem. Lhp1p is also required for efficient aminoacylation of two wild-type tRNAs when yeast are grown at low temperature. These experiments reveal that pre-tRNAs can require protein assistance for efficient folding in vivo.

Autoantigens↗

PubMed2003

A lupus-like syndrome develops in mice lacking the Ro 60-kDa protein, a major lupus autoantigen.

Antibodies against a conserved RNA-binding protein, the Ro 60-kDa autoantigen, occur in 24-60% of all patients with systemic lupus erythematosus. Anti-Ro antibodies are correlated with photosensitivity and cutaneous lesions in these patients and with neonatal lupus, a syndrome in which mothers with anti-Ro antibodies give birth to children with complete congenital heart block and photosensitive skin lesions. In higher eukaryotes, the Ro protein binds small RNAs of unknown function known as Y RNAs. Because the Ro protein also binds misfolded 5S rRNA precursors, it is proposed to function in a quality-control pathway for ribosome biogenesis. Consistent with a role in the recognition or repair of intracellular damage, an orthologue of Ro in the radiation-resistant eubacterium Deinococcus radiodurans contributes to survival of this bacterium after UV irradiation. Here, we show that mice lacking the Ro protein develop an autoimmune syndrome characterized by anti-ribosome antibodies, anti-chromatin antibodies, and glomerulonephritis. Moreover, in one strain background, Ro-/- mice display increased sensitivity to irradiation with UV light. Thus, one function of this major human autoantigen may be to protect against autoantibody development, possibly by sequestering defective ribonucleoproteins from immune surveillance. Furthermore, the finding that mice lacking the Ro protein are photosensitive suggests that loss of Ro function could contribute to the photosensitivity associated with anti-Ro antibodies in humans.

Animals↗

PubMed2001

The La protein.

Ubiquitous in eukaryotic cells, the La protein associates with the 3' termini of many newly synthesized small RNAs. RNAs bound by the La protein include all nascent transcripts made by RNA polymerase III as well as certain small RNAs synthesized by other RNA polymerases. Recent genetic and biochemical analyses have revealed that binding by the La protein protects the 3' ends of these RNAs from exonucleases. This La-mediated stabilization is required for the normal pathway of pre-tRNA maturation, facilitates assembly of small RNAs into functional RNA-protein complexes, and contributes to nuclear retention of certain small RNAs. Studies of mutant La proteins have given some insights into how the La protein specifically recognizes its RNA targets. However, many questions remain regarding the molecular mechanisms by which La protein binding influences multiple steps in small RNA biogenesis. This review focuses on the roles of the La protein in small RNA biogenesis and also discusses data that implicate the La protein in the translation of specific mRNAs.

Adenosine Triphosphatases↗