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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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Domain mapping of the RNA chaperone activity of human La.A, architecture of the human La protein and the deletion mutants used in this study. NRE, nuclear retention element; SBM, short basic motif; NLS, nuclear localization signal. B, kann1, kS.D., and kann2 rates for indicated mutants of the La domain at 100 nm (saturating concentration for wild-type hLa). C, kann1, kS.D. and kann2 rates for indicated mutants of the La domain at 5× concentration (500 nm).
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Figure 3: Domain mapping of the RNA chaperone activity of human La.A, architecture of the human La protein and the deletion mutants used in this study. NRE, nuclear retention element; SBM, short basic motif; NLS, nuclear localization signal. B, kann1, kS.D., and kann2 rates for indicated mutants of the La domain at 100 nm (saturating concentration for wild-type hLa). C, kann1, kS.D. and kann2 rates for indicated mutants of the La domain at 5× concentration (500 nm).

Mentions: We performed strand annealing and dissociation assays using various point and deletion mutants of hLa to identify which elements were required for each activity (Fig. 3 and 4). Surprisingly, we found that both the N- and C-terminal halves of hLa (hLa 1–235 and hLa 225–408) harbored both strand annealing and strand dissociation activity, although neither had strand annealing activity at the same level as full-length hLa (hLa 1–235 kann1 = 0.025 s−1 and hLa 225–408 = 0.021 s−1versus hLa kann1 = 0.058 s−1; Fig. 3B). Because the C-terminal half of human La is not conserved and is largely absent in La proteins from fission and budding yeast (see “Discussion”), we focused on studying the N-terminal La domain shared between all La homologs to gain insight into how La-dependent RNA chaperone activity should function generally across eukaryotes. This N-terminal region includes the winged helix fold containing La motif followed by RRM1, which in addition to having the expected β1α1β2β3α2β4 RRM fold (39) contains an extra C-terminal α-helix (α3) not typically found in unrelated RRMs and previously hypothesized to be in an orientation particular to La proteins (Fig. 4A) (9). Notably, this helix includes three basic lysine residues, two of which point toward the canonical RNA binding RRM1 β-sheet surface, as well as a universally conserved aromatic residue (hLa: Tyr-188) that stacks upon a conserved aromatic (hLa: Tyr-114) also on this β-sheet (39). We found that the La motif in isolation (amino acids 1–104) had insignificant strand annealing or dissociation activity compared with controls (Fig. 3B). Surprisingly, we found that including both the La motif and the canonical β1α1β2β3α2β4 RRM fold (hLa 1–187) also showed very little activity, suggesting that the region between hLa 187 and 235 is important for RNA chaperone activity. This region includes the noncanonical α3-helix as well as a predicted unstructured linker region between RRM1 and RRM2.


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

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

Domain mapping of the RNA chaperone activity of human La.A, architecture of the human La protein and the deletion mutants used in this study. NRE, nuclear retention element; SBM, short basic motif; NLS, nuclear localization signal. B, kann1, kS.D., and kann2 rates for indicated mutants of the La domain at 100 nm (saturating concentration for wild-type hLa). C, kann1, kS.D. and kann2 rates for indicated mutants of the La domain at 5× concentration (500 nm).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Domain mapping of the RNA chaperone activity of human La.A, architecture of the human La protein and the deletion mutants used in this study. NRE, nuclear retention element; SBM, short basic motif; NLS, nuclear localization signal. B, kann1, kS.D., and kann2 rates for indicated mutants of the La domain at 100 nm (saturating concentration for wild-type hLa). C, kann1, kS.D. and kann2 rates for indicated mutants of the La domain at 5× concentration (500 nm).
Mentions: We performed strand annealing and dissociation assays using various point and deletion mutants of hLa to identify which elements were required for each activity (Fig. 3 and 4). Surprisingly, we found that both the N- and C-terminal halves of hLa (hLa 1–235 and hLa 225–408) harbored both strand annealing and strand dissociation activity, although neither had strand annealing activity at the same level as full-length hLa (hLa 1–235 kann1 = 0.025 s−1 and hLa 225–408 = 0.021 s−1versus hLa kann1 = 0.058 s−1; Fig. 3B). Because the C-terminal half of human La is not conserved and is largely absent in La proteins from fission and budding yeast (see “Discussion”), we focused on studying the N-terminal La domain shared between all La homologs to gain insight into how La-dependent RNA chaperone activity should function generally across eukaryotes. This N-terminal region includes the winged helix fold containing La motif followed by RRM1, which in addition to having the expected β1α1β2β3α2β4 RRM fold (39) contains an extra C-terminal α-helix (α3) not typically found in unrelated RRMs and previously hypothesized to be in an orientation particular to La proteins (Fig. 4A) (9). Notably, this helix includes three basic lysine residues, two of which point toward the canonical RNA binding RRM1 β-sheet surface, as well as a universally conserved aromatic residue (hLa: Tyr-188) that stacks upon a conserved aromatic (hLa: Tyr-114) also on this β-sheet (39). We found that the La motif in isolation (amino acids 1–104) had insignificant strand annealing or dissociation activity compared with controls (Fig. 3B). Surprisingly, we found that including both the La motif and the canonical β1α1β2β3α2β4 RRM fold (hLa 1–187) also showed very little activity, suggesting that the region between hLa 187 and 235 is important for RNA chaperone activity. This region includes the noncanonical α3-helix as well as a predicted unstructured linker region between RRM1 and RRM2.

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