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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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hLa exhibits strong preference for pre-tRNA over a UUU-3'OH trailer. (a & b) Binding reactions contained constant amounts of hLa protein (100 nM) and a trace amount of 32P-RNA (~0.1 nM, pre-tRNA in A, or UUU-3'OH trailer in (a), and varying amounts of unlabelled (cold) competitor RNAs as indicated above the lanes and described in the text, in 1 mM Mg2+. The data in A and B were quantified and analyzed in (c) and (d) respectively.
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Figure 3: hLa exhibits strong preference for pre-tRNA over a UUU-3'OH trailer. (a & b) Binding reactions contained constant amounts of hLa protein (100 nM) and a trace amount of 32P-RNA (~0.1 nM, pre-tRNA in A, or UUU-3'OH trailer in (a), and varying amounts of unlabelled (cold) competitor RNAs as indicated above the lanes and described in the text, in 1 mM Mg2+. The data in A and B were quantified and analyzed in (c) and (d) respectively.

Mentions: For Method 2, analyses focus on RNA concentrations around the Kd obtained by method 1. For Method 2, varying amounts of unlabeled RNA were added to constant amounts of 32P-RNA and La33. Bound and free 32P-RNA fractions were quantified and Kd determined by Scatchard analysis. EMSAs were performed in triplicate. Method 2 supports a greater amount of data, is independent of the fraction of active protein, and analysis includes quality assurance parameters33. Results for 0 and 10 mM Mg2+ are shown in Fig. 2A and B, respectively and summarized in Fig. 2G (also see Supplementary Fig. 2 & Fig 3). The Kd of 13.6 nM that we obtained for the 12 nt UUU-3'OH trailer in 10 mM Mg2+ is similar to 7.1 nM and 25 nM obtained by others in the presence of Mg2+ for similar ligands4,34. Our Kd of 2.4 nM for pre-tRNAArg is similar to 7.3 nM for pre-tRNAVal, in the absence of Mg2+35.


Precursor-product discrimination by La protein during tRNA metabolism.

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

hLa exhibits strong preference for pre-tRNA over a UUU-3'OH trailer. (a & b) Binding reactions contained constant amounts of hLa protein (100 nM) and a trace amount of 32P-RNA (~0.1 nM, pre-tRNA in A, or UUU-3'OH trailer in (a), and varying amounts of unlabelled (cold) competitor RNAs as indicated above the lanes and described in the text, in 1 mM Mg2+. The data in A and B were quantified and analyzed in (c) and (d) respectively.
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Related In: Results  -  Collection

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Figure 3: hLa exhibits strong preference for pre-tRNA over a UUU-3'OH trailer. (a & b) Binding reactions contained constant amounts of hLa protein (100 nM) and a trace amount of 32P-RNA (~0.1 nM, pre-tRNA in A, or UUU-3'OH trailer in (a), and varying amounts of unlabelled (cold) competitor RNAs as indicated above the lanes and described in the text, in 1 mM Mg2+. The data in A and B were quantified and analyzed in (c) and (d) respectively.
Mentions: For Method 2, analyses focus on RNA concentrations around the Kd obtained by method 1. For Method 2, varying amounts of unlabeled RNA were added to constant amounts of 32P-RNA and La33. Bound and free 32P-RNA fractions were quantified and Kd determined by Scatchard analysis. EMSAs were performed in triplicate. Method 2 supports a greater amount of data, is independent of the fraction of active protein, and analysis includes quality assurance parameters33. Results for 0 and 10 mM Mg2+ are shown in Fig. 2A and B, respectively and summarized in Fig. 2G (also see Supplementary Fig. 2 & Fig 3). The Kd of 13.6 nM that we obtained for the 12 nt UUU-3'OH trailer in 10 mM Mg2+ is similar to 7.1 nM and 25 nM obtained by others in the presence of Mg2+ for similar ligands4,34. Our Kd of 2.4 nM for pre-tRNAArg is similar to 7.3 nM for pre-tRNAVal, in the absence of Mg2+35.

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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