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RNA chaperone activity of human La protein is mediated by variant RNA recognition motif.

ABSTRACT: La proteins are conserved factors in eukaryotes that bind and protect the 3' trailers of pre-tRNAs from exonuclease digestion via sequence-specific recognition of UUU-3'OH. La has also been hypothesized to assist pre-tRNAs in attaining their native fold through RNA chaperone activity. In addition to binding polymerase III transcripts, human La has also been shown to enhance the translation of several internal ribosome entry sites and upstream ORF-containing mRNA targets, also potentially through RNA chaperone activity. Using in vitro FRET-based assays, we show that human and Schizosaccharomyces pombe La proteins harbor RNA chaperone activity by enhancing RNA strand annealing and strand dissociation. We use various RNA substrates and La mutants to show that UUU-3'OH-dependent La-RNA binding is not required for this function, and we map RNA chaperone activity to its RRM1 motif including a noncanonical α3-helix. We validate the importance of this α3-helix by appending it to the RRM of the unrelated U1A protein and show that this fusion protein acquires significant strand annealing activity. Finally, we show that residues required for La-mediated RNA chaperone activity in vitro are required for La-dependent rescue of tRNA-mediated suppression via a mutated suppressor tRNA in vivo. This work delineates the structural elements required for La-mediated RNA chaperone activity and provides a basis for understanding how La can enhance the folding of its various RNA targets.

SUBMITTER: Naeeni AR

PROVIDER: S-EPMC3285324 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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RNA chaperone activity of human La protein is mediated by variant RNA recognition motif.

Naeeni Amir R AR Conte Maria R MR Bayfield Mark A MA

The Journal of biological chemistry 20111227 8

La proteins are conserved factors in eukaryotes that bind and protect the 3' trailers of pre-tRNAs from exonuclease digestion via sequence-specific recognition of UUU-3'OH. La has also been hypothesized to assist pre-tRNAs in attaining their native fold through RNA chaperone activity. In addition to binding polymerase III transcripts, human La has also been shown to enhance the translation of several internal ribosome entry sites and upstream ORF-containing mRNA targets, also potentially through ...[more]

PMID: 22203678

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Project description:Systemic lupus erythematosus (SLE) is characterized by the production of grouped sets of autoantibodies targeting mainly the U1 ribonucleoprotein (RNP) and/or Ro/La RNP particles. Intraparticle diversification of the autoimmune response is believed to occur via epitope spreading. So far, it is not known how the autoimmune response "jumps" from one particle to another. To the extent that the majority of nuclear autoantigens in SLE are RNA binding proteins and major epitopes were previously mapped within their RRM (RNA recognition motifs), conserved sequences within RRM could be involved in the intermolecular and inter-particle diversification process of the autoimmune response. We investigated the potential of RRM of the La/SSB autoantigen to induce antibodies that cross-recognize components of the U1-RNP particle and therefore its capacity to produce interparticle epitope spreading. We immunized New Zealand white rabbits with a peptide corresponding to the epitope 145-164 of La/SSB (belonging to the RRM of La/SSB), attached in four copies on a scaffold carrier. Sera were drawn from 20 sera of patients with SLE and anti-U1-RNP antibodies and 26 sera of primary Sjögren syndrome patients with anti-La/SSB antibodies. All sera were evaluated for reactivity against the major epitope of La/SSB (pep349-364), the RNP antigen and the RRM-related epitope of La/SSB (pep145-164). Specific antibodies against pep145-164 were purified with immunoaffinity columns from selected sera. After the immunization of the animals with pep145-164, a specific IgG antibody response was detected, directed against the La/SSB autoantigen (wks 3-7), the immunizing peptide (wks 3-27), and the RNP autoantigen (wks 7-20). This response gradually decreased to low levels between postimmunization wks 27-42. Purified antibodies against pep145-164 recognized La/SSB and a 70-kD autoantigen in Western blot and exhibited significant reactivity in anti-U1-RNP ELISA. Depletion of anti-pep145-164 antibodies eliminated anti-U1-RNP reactivity from immunized rabbit sera but not from human sera. In addition, pep145-164 was recognized to a greater extent by autoimmune sera with anti-RNP reactivity compared with anti-La/SSB-positive sera, in contrast to pep349-364 of La/SSB, which was recognized almost exclusively by sera with anti-La/SSB reactivity. These data suggest that the RRM region of La/SSB can trigger interparticle B-cell diversification to U1-RNP-70 autoantigen via molecular mimicry. Identification of key sequences that trigger and perpetuate the autoimmune process is particularly important for understanding pathogenetic mechanisms in autoimmunity.

Dataset Information

RNA chaperone activity of human La protein is mediated by variant RNA recognition motif.

Publications

RNA chaperone activity of human La protein is mediated by variant RNA recognition motif.

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