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

Naeeni AR, Conte MR, Bayfield MA - J. Biol. Chem. (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada.

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.

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hLa and Sla1p display RNA strand association and strand dissociation activity.A, assay for strand annealing and strand dissociation. B, RNA strand annealing (phase I) and strand dissociation (phase II) of hLa compared with StpA was measured as a change in FRET index between complementary Cy5- and Cy3-labeled RNA strands over time. C, FRET index data over time were used to calculate strand annealing rates (kann1, phase I), strand dissociation rates (kS.D., phase II) and strand annealing in phase II in the absence of strand dissociation (kann2, phase II). The error bars show the standard deviation of at least three separate experiments.
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Figure 1: hLa and Sla1p display RNA strand association and strand dissociation activity.A, assay for strand annealing and strand dissociation. B, RNA strand annealing (phase I) and strand dissociation (phase II) of hLa compared with StpA was measured as a change in FRET index between complementary Cy5- and Cy3-labeled RNA strands over time. C, FRET index data over time were used to calculate strand annealing rates (kann1, phase I), strand dissociation rates (kS.D., phase II) and strand annealing in phase II in the absence of strand dissociation (kann2, phase II). The error bars show the standard deviation of at least three separate experiments.

Mentions: To investigate strand annealing and strand dissociation activities of La proteins, we performed FRET-based assays that rely on the annealing of 21-nucleotide complementary RNA strands labeled at their 5′ ends with the fluorophores Cy5 (top strand) or Cy3 (bottom strand) (35, 37) (Fig. 1A). Briefly, the RNA substrates were incubated together in a heated fluorimeter cuvette held at a constant temperature of 37 °C, and the rate of strand annealing (kann1) in the presence or absence of an RNA chaperone was measured as an increase in FRET index between the fluorophores over time (phase I; Fig. 1B). In phase II (at t = 180 s), the capacity of candidate RNA chaperones to dissociate annealed duplexes formed in phase I was determined by injection of an excess of unlabeled bottom strand. In the absence of an RNA chaperone with strand dissociation activity, this results in an increase in measured strand annealing (kann2) because the concentration of one of the partners in the annealing reaction has now increased (37). However, in the presence of an RNA chaperone with strand dissociation activity, the excess unlabeled bottom strands can trap dissociated upper strands resulting in a decrease in FRET index (Fig. 1B; measured as the rate of strand dissociation, kS.D.). As negative controls, we measured the rates of strand annealing and strand dissociation in the absence of added protein or in the presence of BSA. As a positive control, we performed assays using the E. coli StpA protein, which has been previously characterized as harboring both strand annealing and dissociation activity (37).


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

Naeeni AR, Conte MR, Bayfield MA - J. Biol. Chem. (2011)

hLa and Sla1p display RNA strand association and strand dissociation activity.A, assay for strand annealing and strand dissociation. B, RNA strand annealing (phase I) and strand dissociation (phase II) of hLa compared with StpA was measured as a change in FRET index between complementary Cy5- and Cy3-labeled RNA strands over time. C, FRET index data over time were used to calculate strand annealing rates (kann1, phase I), strand dissociation rates (kS.D., phase II) and strand annealing in phase II in the absence of strand dissociation (kann2, phase II). The error bars show the standard deviation of at least three separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285324&req=5

Figure 1: hLa and Sla1p display RNA strand association and strand dissociation activity.A, assay for strand annealing and strand dissociation. B, RNA strand annealing (phase I) and strand dissociation (phase II) of hLa compared with StpA was measured as a change in FRET index between complementary Cy5- and Cy3-labeled RNA strands over time. C, FRET index data over time were used to calculate strand annealing rates (kann1, phase I), strand dissociation rates (kS.D., phase II) and strand annealing in phase II in the absence of strand dissociation (kann2, phase II). The error bars show the standard deviation of at least three separate experiments.
Mentions: To investigate strand annealing and strand dissociation activities of La proteins, we performed FRET-based assays that rely on the annealing of 21-nucleotide complementary RNA strands labeled at their 5′ ends with the fluorophores Cy5 (top strand) or Cy3 (bottom strand) (35, 37) (Fig. 1A). Briefly, the RNA substrates were incubated together in a heated fluorimeter cuvette held at a constant temperature of 37 °C, and the rate of strand annealing (kann1) in the presence or absence of an RNA chaperone was measured as an increase in FRET index between the fluorophores over time (phase I; Fig. 1B). In phase II (at t = 180 s), the capacity of candidate RNA chaperones to dissociate annealed duplexes formed in phase I was determined by injection of an excess of unlabeled bottom strand. In the absence of an RNA chaperone with strand dissociation activity, this results in an increase in measured strand annealing (kann2) because the concentration of one of the partners in the annealing reaction has now increased (37). However, in the presence of an RNA chaperone with strand dissociation activity, the excess unlabeled bottom strands can trap dissociated upper strands resulting in a decrease in FRET index (Fig. 1B; measured as the rate of strand dissociation, kS.D.). As negative controls, we measured the rates of strand annealing and strand dissociation in the absence of added protein or in the presence of BSA. As a positive control, we performed assays using the E. coli StpA protein, which has been previously characterized as harboring both strand annealing and dissociation activity (37).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada.

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.

Show MeSH
Related in: MedlinePlus