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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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The hLa loop mutant is defective in tRNA maturation in vivo. (a) tRNA-mediated suppression (TMS) activity was assayed in S. pombe; pRep is the empty vector; all other hLa constructs indicated were cloned in pRep17,18,36,38. (b) Northern blot analysis of RNAs extracted from cells in A above. Upper panel shows blot probed for intron-containing pre-tRNALysCUU species, which migrate as upper, middle and lower bands (see text). Lower panel shows the same blot reprobed for U5 snRNA, which served as a loading control for quantitation, normalized to hLa = 1.0, lane 3. (c) RNA chaperone assays were performed in vitro using the cis-splicing intron RNA23. Activity is reflected by a decrease in unspliced RNA (ln U/Uo, Experimental Procedures) for each of the proteins indicated. Error bars reflect triplicate samples for each point. (d) Coomassie blue stained gel of the BSA (lane 1), hLa (lane 2) and hLa-loop (lane 3) proteins used in the assay.
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Figure 5: The hLa loop mutant is defective in tRNA maturation in vivo. (a) tRNA-mediated suppression (TMS) activity was assayed in S. pombe; pRep is the empty vector; all other hLa constructs indicated were cloned in pRep17,18,36,38. (b) Northern blot analysis of RNAs extracted from cells in A above. Upper panel shows blot probed for intron-containing pre-tRNALysCUU species, which migrate as upper, middle and lower bands (see text). Lower panel shows the same blot reprobed for U5 snRNA, which served as a loading control for quantitation, normalized to hLa = 1.0, lane 3. (c) RNA chaperone assays were performed in vitro using the cis-splicing intron RNA23. Activity is reflected by a decrease in unspliced RNA (ln U/Uo, Experimental Procedures) for each of the proteins indicated. Error bars reflect triplicate samples for each point. (d) Coomassie blue stained gel of the BSA (lane 1), hLa (lane 2) and hLa-loop (lane 3) proteins used in the assay.

Mentions: We next examined the hLa-loop mutant for tRNA maturation in S. pombe as monitored by tRNA-mediated suppression (TMS) of a premature stop codon in ade6-704, which alleviates the accumulation of red pigment. By this assay, hLa can replace the function of S. pombe La protein (Sla1p) in vivo17, 18. We previously characterized suppressor tRNA alleles that vary in dependence on La, Rrp6 (3' exonuclease component of the exosome), and the pol III termination subunit Rpc11p (a pol III-associated 3'-5' exonuclease), for efficient maturation18,31. Mutation of the conserved aromatic residues, Y114 and F155, on the RNP-1 and -2 motifs of the RRM1 β-surface compromised maturation of the structurally-defective suppressor pre-tRNA in the strain, ySK5, but had less effect in ySH9, which contains a wild-type suppressor tRNA18. For the present study, analysis was limited to the wild-type suppressor tRNA in ySH9 (Fig. 5A–B). Consistent with prior data, hLa was more active for TMS than the negative controls pRep vector and hLa26-408 which lacks residues involved in UUU-3'OH recognition17,18,36 (Fig 5A, sectors 1 & 2). hLa-Y114A/F155A also served as a control (sector 3). hLaloop-3 was less active than hLa (Fig. 5A, compare sectors 3 & 5), indicating that one or more of the basic residues in loop-3 that mediate UUU-OH-independent pre-tRNA binding contribute to functional tRNA maturation in vivo. We also combined the RRM1 loop-3 and the β-sheet mutations. These mutants, hLaY114A-loop and hLaY114A/F155A-loop were increasingly compromised for TMS (Fig. 5A, sectors 6 & 7). As shown below, these RRM1 mutants maintain pre-tRNA UUU-OH 3' end protection activity.


Precursor-product discrimination by La protein during tRNA metabolism.

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

The hLa loop mutant is defective in tRNA maturation in vivo. (a) tRNA-mediated suppression (TMS) activity was assayed in S. pombe; pRep is the empty vector; all other hLa constructs indicated were cloned in pRep17,18,36,38. (b) Northern blot analysis of RNAs extracted from cells in A above. Upper panel shows blot probed for intron-containing pre-tRNALysCUU species, which migrate as upper, middle and lower bands (see text). Lower panel shows the same blot reprobed for U5 snRNA, which served as a loading control for quantitation, normalized to hLa = 1.0, lane 3. (c) RNA chaperone assays were performed in vitro using the cis-splicing intron RNA23. Activity is reflected by a decrease in unspliced RNA (ln U/Uo, Experimental Procedures) for each of the proteins indicated. Error bars reflect triplicate samples for each point. (d) Coomassie blue stained gel of the BSA (lane 1), hLa (lane 2) and hLa-loop (lane 3) proteins used in the assay.
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Figure 5: The hLa loop mutant is defective in tRNA maturation in vivo. (a) tRNA-mediated suppression (TMS) activity was assayed in S. pombe; pRep is the empty vector; all other hLa constructs indicated were cloned in pRep17,18,36,38. (b) Northern blot analysis of RNAs extracted from cells in A above. Upper panel shows blot probed for intron-containing pre-tRNALysCUU species, which migrate as upper, middle and lower bands (see text). Lower panel shows the same blot reprobed for U5 snRNA, which served as a loading control for quantitation, normalized to hLa = 1.0, lane 3. (c) RNA chaperone assays were performed in vitro using the cis-splicing intron RNA23. Activity is reflected by a decrease in unspliced RNA (ln U/Uo, Experimental Procedures) for each of the proteins indicated. Error bars reflect triplicate samples for each point. (d) Coomassie blue stained gel of the BSA (lane 1), hLa (lane 2) and hLa-loop (lane 3) proteins used in the assay.
Mentions: We next examined the hLa-loop mutant for tRNA maturation in S. pombe as monitored by tRNA-mediated suppression (TMS) of a premature stop codon in ade6-704, which alleviates the accumulation of red pigment. By this assay, hLa can replace the function of S. pombe La protein (Sla1p) in vivo17, 18. We previously characterized suppressor tRNA alleles that vary in dependence on La, Rrp6 (3' exonuclease component of the exosome), and the pol III termination subunit Rpc11p (a pol III-associated 3'-5' exonuclease), for efficient maturation18,31. Mutation of the conserved aromatic residues, Y114 and F155, on the RNP-1 and -2 motifs of the RRM1 β-surface compromised maturation of the structurally-defective suppressor pre-tRNA in the strain, ySK5, but had less effect in ySH9, which contains a wild-type suppressor tRNA18. For the present study, analysis was limited to the wild-type suppressor tRNA in ySH9 (Fig. 5A–B). Consistent with prior data, hLa was more active for TMS than the negative controls pRep vector and hLa26-408 which lacks residues involved in UUU-3'OH recognition17,18,36 (Fig 5A, sectors 1 & 2). hLa-Y114A/F155A also served as a control (sector 3). hLaloop-3 was less active than hLa (Fig. 5A, compare sectors 3 & 5), indicating that one or more of the basic residues in loop-3 that mediate UUU-OH-independent pre-tRNA binding contribute to functional tRNA maturation in vivo. We also combined the RRM1 loop-3 and the β-sheet mutations. These mutants, hLaY114A-loop and hLaY114A/F155A-loop were increasingly compromised for TMS (Fig. 5A, sectors 6 & 7). As shown below, these RRM1 mutants maintain pre-tRNA UUU-OH 3' end protection activity.

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
Related in: MedlinePlus