The small nuclear ribonucleoprotein SS-B/La binds RNA with a conserved protease-resistant domain of 28 kilodaltons.

Chan, Edward K. L.; Tan, Eng M.

doi:10.1128/mcb.7.7.2588

Cited by 26 publications

(20 citation statements)

References 30 publications

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“…This assay was used to show that phosphorylated and unphosphorylated La proteins exhibit differential transcription factor activities (15). It was unclear how phosphorylation at a single site, serine 366, in the C terminus of La could regulate transcription factor activity, since the only known transcription-related activity of La, i.e., nascent-transcript binding, had been localized to the N-terminal domain (7). The results presented here help clarify this issue (see Discussion).…”

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confidence: 71%

A Carboxy-Terminal Basic Region Controls RNA Polymerase III Transcription Factor Activity of Human La Protein

Goodier

Fan

Maraia

1997

Molecular and Cellular Biology

101

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). Others have previously dissected the La protein into an N-terminal domain that binds RNA and a C-terminal domain that does not. Here, deletion and substitution mutants of La were examined for general RNA binding, RNA 3-end protection, and transcription factor activity. Although some La mutants altered in a C-terminal basic region bind RNA in mobility shift assays, they are defective in RNA 3-end protection and do not support transcription, while one C-terminal substitution mutant is defective only in transcription. Moreover, a Cterminal fragment lacking RNA binding activity appears able to support low levels of transcription by pol III. While efficient multiround transcription is supported only by mutants that bind RNA and contain a C-terminal basic region. These analyses indicate that RNA binding contributes to but is not sufficient for La transcription factor activity and that the C-terminal domain plays a role in transcription that is distinguishable from simple RNA binding. The transcription factor activity of La can be reversibly inhibited by RNA, suggesting the potential for feedback inhibition of pol III transcription.Eukaryotic RNA polymerase III (pol III) is responsible for synthesizing the abundant transcripts of tRNA, 5S rRNA, 7SL RNA, and U6 RNA genes as well as other small RNA genes (reviewed in reference 44). To produce a sufficient quantity of RNAs of the correct structure, transcription initiation and termination must be accurate and reinitiation must be efficient. pol III alone cannot accomplish this and must rely on transcription factors (TFs) that bind to control elements and direct RNA synthesis. The adenovirus-associated (VA1) RNA gene and cellular tRNA and Alu genes have internal promoters and 3Ј terminators that direct transcription by pol III. Mammalian TFIIIC1 and TFIIIC2 bind to the control elements of these genes (12, 43). After TFIIIB joins, the resulting preinitiation complex is stable and can be recycled for multiple rounds of transcription by pol III (5,21,27,43).Mammalian in vitro transcription assays have used reconstituted systems that contain at least one partially purified fraction. For example, mammalian TFIIIC2, TFIIIB, and pol III have been highly purified, while TFIIIC1 and TFIIIC1Ј represent relatively crude yet essential fractions (42, 43). In the Saccharomyces cerevisiae system, elegant studies have shown that recombinant or highly purified TFIIIB and TFIIIC are sufficient for multiround transcription (21), although it has also been reported that transcription can be stimulated by TFIIIE, a partially characterized fraction (13, 37). Thus, in both yeast and mammalian pol III systems, while the central factors required for in vitro transcription have been identified and continue to be characterized, evidence also suggests that ancillary, less well characterized factors may stimulate transcription. While remarkable progress has been made in understanding assembly of the preinitiation complex, relatively less effort has been directed toward understanding of the mechanism...

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confidence: 71%

A Carboxy-Terminal Basic Region Controls RNA Polymerase III Transcription Factor Activity of Human La Protein

Goodier

Fan

Maraia

1997

Molecular and Cellular Biology

101

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“…In previous studies, the antigenic regions of La/SSB were detected by molecular biological techniques. In fact, Chan et al [2,3], using controlled proteolytic degradation with Staphylococcus aureus V8 protease, identified two antigenically independent sets of protease-resistant peptides termed X and Y. Sturgess et al, using cDNA encoding 87% of La/ SSB protein, identified a major antigenic epitope within the 103 aa of the C-terminal portion of the protein [4]. Rauh & Lührmann [5] suggested three independent immunodominant regions, spanning the 284-292, 293-345 and 346-383 sequences of La/SSB.…”

Section: Introductionmentioning

confidence: 99%

Fine specificity of autoantibodies to La/SSB: epitope mapping, and characterization

Tzioufas

Yiannaki

Sakarellos‐Daitsiotis

et al. 1997

Clinical and Experimental Immunology

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SUMMARYThe B cell epitope mapping of La/SSB was performed using 20 mer synthetic peptides overlapping by eight amino acids covering the whole sequence of the protein. IgG, purified from sera of five patients with systemic lupus erythematosus (SLE) and four sera from patients with primary Sjögren's syndrome (pSS) were tested against the overlapping synthetic peptides. Peptides highly reactive with purified IgG were those spanning the regions 145-164, 289-308, 301-320 and 349-368 364 . Predicted features and molecular similarities of the defined epitopes were investigated using protein databases. The La epitope 147 HKAFKGSI 154 presented 83·3% similarity with the 139 HKGFKGVD 146 region of human myelin basic protein (MBP) and 72% similarity with the fragment YKNFKGTI of human DNA topoisomerase II. Peptides corresponding to these sequences cross-reacted with anti-La/SSB antibodies. Sixty-three sera with anti-La/SSB antibodies from patients with pSS or SLE, 35 sera without anti-La/SSB antibodies from patients with SS or SLE and 41 sera from age/sex-matched healthy blood donors were tested against biotinylated synthetic epitope analogues in order to determine their sensitivity and specificity for the detection of anti-La/SSB antibodies. Anti-La/SSB were detected with various frequencies ranging from 20% to epitope 147 HKAFKGSI 154 to 100% to epitope 349 GSGKGKVQGKKTKF 364 . The overall sensitivity and specificity using all assays with the synthetic peptides were found to be 93·6% and 85·6%, respectively. In conclusion, antibodies to La/SSB constitute a heterogeneous population, directed against different linear B cell epitopes of the molecule. The epitope 147 HKAFKGSI 154 presents molecular similarity with fragments of two other autoantigens, i.e. human MBP and DNA topoisomerase II. Finally, synthetic epitope analogues exhibit high sensitivity and specificity for the detection of anti-La/SSB antibodies.

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“…It was first discovered as an autoantigen recognized by antibodies present in sera of patients suffering from systemic lupus erythematosus and Sjögren's syndrome (1,2). The La protein is a member of a large group of RNA-binding proteins containing RNA recognition motifs (RRM) 1 (3)(4)(5)(6)(7)(8) and is implicated in several steps of RNA metabolism. Among the different La proteins identified in a variety of organisms, the N-terminal part is highly conserved (9).…”

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Molecular Characterization of the Human La Protein·Hepatitis B Virus RNA.B Interaction in Vitro

Horke

Reumann

Rang³

et al. 2002

Journal of Biological Chemistry

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The La protein was recently identified as a host factor potentially involved in the cytokine-induced post-transcriptional down-regulation of hepatitis B virus (HBV) RNA. The La binding site was mapped to a predicted stem-loop structure within a region shared by all HBV RNAs, and it was concluded that the La protein might be an HBV RNA-stabilizing factor. To characterize the RNA binding mediated by the different RNA recognition motifs (RRMs) of the human La protein, several La deletion mutants were produced and analyzed for HBV RNA binding ability. The data demonstrate that the first RRM is not required for binding, whereas the RNP-1 and RNP-2 consensus sequences of the RRM-2 and RRM-3 are separately required for binding, indicating a cooperative function of these two RRMs. Furthermore, the results suggest that multimeric La disassembles into monomeric La upon binding of HBV RNA.B. By gel retardation assay the affinity of the wild type human La⅐HBV RNA.B interaction was determined in the nanomolar range, comparable to the affinity determined for the mouse La⅐HBV RNA.B interaction. This study identified small regions within the human La protein mediating the binding of HBV RNA. Hence, these binding sites might represent targets for novel antiviral strategies based on the disruption of the human La⅐HBV RNA interaction, thereby leading to HBV RNA degradation.The human La protein is a 47-kDa phosphoprotein predominantly localized in the nucleus. It was first discovered as an autoantigen recognized by antibodies present in sera of patients suffering from systemic lupus erythematosus and Sjögren's syndrome (1, 2). The La protein is a member of a large group of RNA-binding proteins containing RNA recognition motifs (RRM) 1 (3-8) and is implicated in several steps of RNA metabolism. Among the different La proteins identified in a variety of organisms, the N-terminal part is highly conserved (9). La was shown to co-immunoprecipitate with a number of small RNA molecules (10). A role for La in the termination of RNA polymerase III transcription has been described. It was shown that La interacts with RNA polymerase III transcripts such as pre-tRNA by binding to a small stretch of uridines at the 3Ј-end common to these transcripts and might be necessary for proper processing of these precursors (11-17). In addition, La is known to interact with a variety of viral and other cellular . La is also suggested to be involved in the capindependent translation initiation of several viruses, including polio virus and hepatitis C virus (19,(27)(28)(29), and more recently evidence is growing that La stabilizes various RNAs, such as histone and hepatitis C and B virus RNA (22,23,25,30,31). At this time point it is not clear yet how La fulfills all of these different functions, however, assuming that this protein acts as a RNA chaperone, thereby stabilizing RNA structures, a function in these varied processes might be envisaged.The human La protein contains three RNA recognition motifs (RRM) involved in the binding of RNAs (9), although t...

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The small nuclear ribonucleoprotein SS-B/La binds RNA with a conserved protease-resistant domain of 28 kilodaltons.

Cited by 26 publications

References 30 publications

A Carboxy-Terminal Basic Region Controls RNA Polymerase III Transcription Factor Activity of Human La Protein

A Carboxy-Terminal Basic Region Controls RNA Polymerase III Transcription Factor Activity of Human La Protein

Fine specificity of autoantibodies to La/SSB: epitope mapping, and characterization

Molecular Characterization of the Human La Protein·Hepatitis B Virus RNA.B Interaction in Vitro

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