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Precursor-product discrimination by La protein during tRNA metabolism.

Bayfield MA, Maraia RJ - Nat. Struct. Mol. Biol. (2009)

Bottom Line: RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA, but not for the UUU-3'OH trailer, and impair tRNA maturation in vivo.Accordingly, the RRM1 mutations also impair an RNA chaperone activity of La.The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA.

View Article: PubMed Central - PubMed

Affiliation: Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
La proteins bind pre-tRNAs at their UUU-3'OH ends, facilitating their maturation. Although the mechanism by which La binds pre-tRNA 3' trailers is known, the function of the RNA binding beta-sheet surface of the RNA-recognition motif (RRM1) is unknown. How La dissociates from UUU-3'OH-containing trailers after 3' processing is also unknown. Here we show that La preferentially binds pre-tRNAs over processed tRNAs or 3' trailer products through coupled use of two sites: one on the La motif and another on the RRM1 beta-surface that binds elsewhere on tRNA. Two sites provide stable pre-tRNA binding, whereas the processed tRNA and 3' trailer are released from their single sites relatively fast. RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA, but not for the UUU-3'OH trailer, and impair tRNA maturation in vivo. We propose that RRM1 functions in activities that are more complex than UUU-3'OH binding. Accordingly, the RRM1 mutations also impair an RNA chaperone activity of La. The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA.

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La RRM1 loop-3 mediates UUU-3’OH-independent tRNA binding. (a) Sequence alignment of the β2-loop 3-β3 regions of the RRM1s of the La proteins from seven organisms followed by the homologous regions of U1A and PABP. Asterisks indicate the basic residues conserved in loop 3 of La proteins. RNP1 is indicated47; in hLa this contains F155, indicated by vertical line above the sequence. (b) Structure of the loop-3 side chains relative to the RRM1 β-sheet surface (adopted from Kotik-Kogan et al., PDB accession # 2VON). The loop-3 basic residues mutated for this study are shown in blue. RRM1 is shown in green, with the conserved aromatic residue side chains (Y114 and F155) on β-strands 1 and 3, in cyan.
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Figure 4: La RRM1 loop-3 mediates UUU-3’OH-independent tRNA binding. (a) Sequence alignment of the β2-loop 3-β3 regions of the RRM1s of the La proteins from seven organisms followed by the homologous regions of U1A and PABP. Asterisks indicate the basic residues conserved in loop 3 of La proteins. RNP1 is indicated47; in hLa this contains F155, indicated by vertical line above the sequence. (b) Structure of the loop-3 side chains relative to the RRM1 β-sheet surface (adopted from Kotik-Kogan et al., PDB accession # 2VON). The loop-3 basic residues mutated for this study are shown in blue. RRM1 is shown in green, with the conserved aromatic residue side chains (Y114 and F155) on β-strands 1 and 3, in cyan.

Mentions: The Mg2+-sensitive UUU-3’OH-independent binding activity was mapped to the hLa domain (not shown). To further localize this activity we examined surface charge distribution (see Supplementary Fig S5) and mutated candidate basic surface patches to evaluate their effect on RNA binding. Several different mutated proteins were examined for tRNA binding, and in some cases other non-UUU-3'OH-containing RNAs (not shown, indicated as "other" in Table 1a. RRM1 loop-3 (R143A/R144A/K148A/K151A, referred to as “hLa-loop” mutant below), which connects RRM1 β2 and β3 strands, reduced binding to pre-tRNA and tRNA 4–5 fold relative to hLa, without much decrease in binding to the UUU-3’OH trailer (Table 1b, also see Supplementary Figs S6 and S7). Moreover, the residual tRNA binding observed for hLa-loop was insensitive to Mg2+ (not shown). Decreased tRNA binding observed for the hLa-loop protein is specific since many other mutated proteins did not decrease tRNA binding (Table 1a). The fact that hLa-loop mutated protein exhibited normal UUU-3'OH-mediated binding, which requires LM-RRM1 inter-motif contacts,4,9 indicates that the La domain is not grossly misfolded. We conclude that loop-3 of RRM1 is an important determinant of a UUU-3'OH-independent RNA binding site on La. The basic residues mutated in La-loop-3 are highly conserved in La proteins but not other RRM proteins such as U1A and PABP (Fig. 4A). Loop-3 forms a basic wall on one side of the RRM β-sheet surface (Fig. 4B).


Precursor-product discrimination by La protein during tRNA metabolism.

Bayfield MA, Maraia RJ - Nat. Struct. Mol. Biol. (2009)

La RRM1 loop-3 mediates UUU-3’OH-independent tRNA binding. (a) Sequence alignment of the β2-loop 3-β3 regions of the RRM1s of the La proteins from seven organisms followed by the homologous regions of U1A and PABP. Asterisks indicate the basic residues conserved in loop 3 of La proteins. RNP1 is indicated47; in hLa this contains F155, indicated by vertical line above the sequence. (b) Structure of the loop-3 side chains relative to the RRM1 β-sheet surface (adopted from Kotik-Kogan et al., PDB accession # 2VON). The loop-3 basic residues mutated for this study are shown in blue. RRM1 is shown in green, with the conserved aromatic residue side chains (Y114 and F155) on β-strands 1 and 3, in cyan.
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Related In: Results  -  Collection

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Figure 4: La RRM1 loop-3 mediates UUU-3’OH-independent tRNA binding. (a) Sequence alignment of the β2-loop 3-β3 regions of the RRM1s of the La proteins from seven organisms followed by the homologous regions of U1A and PABP. Asterisks indicate the basic residues conserved in loop 3 of La proteins. RNP1 is indicated47; in hLa this contains F155, indicated by vertical line above the sequence. (b) Structure of the loop-3 side chains relative to the RRM1 β-sheet surface (adopted from Kotik-Kogan et al., PDB accession # 2VON). The loop-3 basic residues mutated for this study are shown in blue. RRM1 is shown in green, with the conserved aromatic residue side chains (Y114 and F155) on β-strands 1 and 3, in cyan.
Mentions: The Mg2+-sensitive UUU-3’OH-independent binding activity was mapped to the hLa domain (not shown). To further localize this activity we examined surface charge distribution (see Supplementary Fig S5) and mutated candidate basic surface patches to evaluate their effect on RNA binding. Several different mutated proteins were examined for tRNA binding, and in some cases other non-UUU-3'OH-containing RNAs (not shown, indicated as "other" in Table 1a. RRM1 loop-3 (R143A/R144A/K148A/K151A, referred to as “hLa-loop” mutant below), which connects RRM1 β2 and β3 strands, reduced binding to pre-tRNA and tRNA 4–5 fold relative to hLa, without much decrease in binding to the UUU-3’OH trailer (Table 1b, also see Supplementary Figs S6 and S7). Moreover, the residual tRNA binding observed for hLa-loop was insensitive to Mg2+ (not shown). Decreased tRNA binding observed for the hLa-loop protein is specific since many other mutated proteins did not decrease tRNA binding (Table 1a). The fact that hLa-loop mutated protein exhibited normal UUU-3'OH-mediated binding, which requires LM-RRM1 inter-motif contacts,4,9 indicates that the La domain is not grossly misfolded. We conclude that loop-3 of RRM1 is an important determinant of a UUU-3'OH-independent RNA binding site on La. The basic residues mutated in La-loop-3 are highly conserved in La proteins but not other RRM proteins such as U1A and PABP (Fig. 4A). Loop-3 forms a basic wall on one side of the RRM β-sheet surface (Fig. 4B).

Bottom Line: RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA, but not for the UUU-3'OH trailer, and impair tRNA maturation in vivo.Accordingly, the RRM1 mutations also impair an RNA chaperone activity of La.The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA.

View Article: PubMed Central - PubMed

Affiliation: Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
La proteins bind pre-tRNAs at their UUU-3'OH ends, facilitating their maturation. Although the mechanism by which La binds pre-tRNA 3' trailers is known, the function of the RNA binding beta-sheet surface of the RNA-recognition motif (RRM1) is unknown. How La dissociates from UUU-3'OH-containing trailers after 3' processing is also unknown. Here we show that La preferentially binds pre-tRNAs over processed tRNAs or 3' trailer products through coupled use of two sites: one on the La motif and another on the RRM1 beta-surface that binds elsewhere on tRNA. Two sites provide stable pre-tRNA binding, whereas the processed tRNA and 3' trailer are released from their single sites relatively fast. RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA, but not for the UUU-3'OH trailer, and impair tRNA maturation in vivo. We propose that RRM1 functions in activities that are more complex than UUU-3'OH binding. Accordingly, the RRM1 mutations also impair an RNA chaperone activity of La. The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA.

Show MeSH