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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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Two distinct RNA binding sites on La together enhance stable binding to pre-tRNA. (a–c) Scatchard analyses of Method 2 EMSAs performed on pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer at 0, 10 and 1 mM Mg2+. Each titration was done at a constant concentration of La with concentrations of RNA varying, although the La concentration differed for each individual Scatchard plot; Kds are provided in each panel next to each ligand and in panel G. (d–f) Analysis of dissociation of hLa from pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer from data derived from EMSAs, was performed and analyzed as described33. Time scales are 0 to 350 seconds. (g) Kds derived from A–C above and the numerical fraction of the Kds at 0 and 10 mM Mg2+ (0 Mg/10 Mg) as indicated. Standard errors derived from triplicate determinations for pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer were 14.7%, 11.7% and 10.9%, respectively in 0 Mg2+, 5.5%, 7.3% and 8.9%, respectively in 10 mM Mg2+, and 7.7%, 5.9% and 13.9% in 1 mM Mg2+. koffs were derived from D–F above using standard calculations33. The t1/2s were then derived from koff. Standard errors for dissociation of pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer were 8.5%, 7.1% and 10.8%, respectively. The units for koff and t1/2 are min−1 and min, respectively.
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Figure 2: Two distinct RNA binding sites on La together enhance stable binding to pre-tRNA. (a–c) Scatchard analyses of Method 2 EMSAs performed on pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer at 0, 10 and 1 mM Mg2+. Each titration was done at a constant concentration of La with concentrations of RNA varying, although the La concentration differed for each individual Scatchard plot; Kds are provided in each panel next to each ligand and in panel G. (d–f) Analysis of dissociation of hLa from pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer from data derived from EMSAs, was performed and analyzed as described33. Time scales are 0 to 350 seconds. (g) Kds derived from A–C above and the numerical fraction of the Kds at 0 and 10 mM Mg2+ (0 Mg/10 Mg) as indicated. Standard errors derived from triplicate determinations for pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer were 14.7%, 11.7% and 10.9%, respectively in 0 Mg2+, 5.5%, 7.3% and 8.9%, respectively in 10 mM Mg2+, and 7.7%, 5.9% and 13.9% in 1 mM Mg2+. koffs were derived from D–F above using standard calculations33. The t1/2s were then derived from koff. Standard errors for dissociation of pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer were 8.5%, 7.1% and 10.8%, respectively. The units for koff and t1/2 are min−1 and min, respectively.

Mentions: To further examine for differential binding we compared dissociation constants (Kd) for three relevant RNA species, pre-tRNAArgACG, tRNAArgACG and the free 12 nt UUU-3'OH trailer, in two extremes of [Mg2+], 0 and 10 mM, as well as 1 mM (Fig. 2A–C). Of note is the absence of divalent cations in the crystal of hLa bound to UUU-3’OH4, and the recognized association of divalent cations with tRNA32. The 85 nt pre-tRNAArgACG used in Fig. 2 lacks a 5' leader but contains the 12 nt UUU-3'OH-containing trailer covalently linked to tRNAArgACG (i.e., as a contiguous T7 transcript), as this is a substrate for RNase Z26, while the 73 nt tRNAArgACG and the 12 nt UUU-3'OH trailer represent cleavage products. We used two EMSA methods, each with reactions containing 0 or 10 mM Mg2+. In Method 1, varying amounts of hLa were added to trace amount of 32P-RNA. This showed that 12 nt trailer binding was no different in 0 and 10 mM Mg2+, while tRNA and pre-tRNA binding were dramatically compromised in 10 mM versus 0 Mg2+ (Supplementary Fig 1, 0 and 10 mM Mg2+). By method 1, Kd is roughly estimated as the La concentration at which 50% of the RNA is shifted. Kd for the pre-tRNA and tRNA were each estimated at 5–10 nM in 0 mM Mg2+, and ~100 nM in 10 mM Mg2+, whereas Kd for the 12 nt trailer was ~20 nM, with little if any difference in 0 versus 10 mM Mg2+. The relatively large effect of Mg2+ on pre-tRNA and tRNA but not on 12 nt trailer was confirmed by EMSA method 2 as described below (Fig 2A–C).


Precursor-product discrimination by La protein during tRNA metabolism.

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

Two distinct RNA binding sites on La together enhance stable binding to pre-tRNA. (a–c) Scatchard analyses of Method 2 EMSAs performed on pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer at 0, 10 and 1 mM Mg2+. Each titration was done at a constant concentration of La with concentrations of RNA varying, although the La concentration differed for each individual Scatchard plot; Kds are provided in each panel next to each ligand and in panel G. (d–f) Analysis of dissociation of hLa from pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer from data derived from EMSAs, was performed and analyzed as described33. Time scales are 0 to 350 seconds. (g) Kds derived from A–C above and the numerical fraction of the Kds at 0 and 10 mM Mg2+ (0 Mg/10 Mg) as indicated. Standard errors derived from triplicate determinations for pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer were 14.7%, 11.7% and 10.9%, respectively in 0 Mg2+, 5.5%, 7.3% and 8.9%, respectively in 10 mM Mg2+, and 7.7%, 5.9% and 13.9% in 1 mM Mg2+. koffs were derived from D–F above using standard calculations33. The t1/2s were then derived from koff. Standard errors for dissociation of pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer were 8.5%, 7.1% and 10.8%, respectively. The units for koff and t1/2 are min−1 and min, respectively.
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Related In: Results  -  Collection

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Figure 2: Two distinct RNA binding sites on La together enhance stable binding to pre-tRNA. (a–c) Scatchard analyses of Method 2 EMSAs performed on pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer at 0, 10 and 1 mM Mg2+. Each titration was done at a constant concentration of La with concentrations of RNA varying, although the La concentration differed for each individual Scatchard plot; Kds are provided in each panel next to each ligand and in panel G. (d–f) Analysis of dissociation of hLa from pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer from data derived from EMSAs, was performed and analyzed as described33. Time scales are 0 to 350 seconds. (g) Kds derived from A–C above and the numerical fraction of the Kds at 0 and 10 mM Mg2+ (0 Mg/10 Mg) as indicated. Standard errors derived from triplicate determinations for pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer were 14.7%, 11.7% and 10.9%, respectively in 0 Mg2+, 5.5%, 7.3% and 8.9%, respectively in 10 mM Mg2+, and 7.7%, 5.9% and 13.9% in 1 mM Mg2+. koffs were derived from D–F above using standard calculations33. The t1/2s were then derived from koff. Standard errors for dissociation of pre-tRNAArgACG, tRNAArgACG and the 12 nt UUU-3'OH trailer were 8.5%, 7.1% and 10.8%, respectively. The units for koff and t1/2 are min−1 and min, respectively.
Mentions: To further examine for differential binding we compared dissociation constants (Kd) for three relevant RNA species, pre-tRNAArgACG, tRNAArgACG and the free 12 nt UUU-3'OH trailer, in two extremes of [Mg2+], 0 and 10 mM, as well as 1 mM (Fig. 2A–C). Of note is the absence of divalent cations in the crystal of hLa bound to UUU-3’OH4, and the recognized association of divalent cations with tRNA32. The 85 nt pre-tRNAArgACG used in Fig. 2 lacks a 5' leader but contains the 12 nt UUU-3'OH-containing trailer covalently linked to tRNAArgACG (i.e., as a contiguous T7 transcript), as this is a substrate for RNase Z26, while the 73 nt tRNAArgACG and the 12 nt UUU-3'OH trailer represent cleavage products. We used two EMSA methods, each with reactions containing 0 or 10 mM Mg2+. In Method 1, varying amounts of hLa were added to trace amount of 32P-RNA. This showed that 12 nt trailer binding was no different in 0 and 10 mM Mg2+, while tRNA and pre-tRNA binding were dramatically compromised in 10 mM versus 0 Mg2+ (Supplementary Fig 1, 0 and 10 mM Mg2+). By method 1, Kd is roughly estimated as the La concentration at which 50% of the RNA is shifted. Kd for the pre-tRNA and tRNA were each estimated at 5–10 nM in 0 mM Mg2+, and ~100 nM in 10 mM Mg2+, whereas Kd for the 12 nt trailer was ~20 nM, with little if any difference in 0 versus 10 mM Mg2+. The relatively large effect of Mg2+ on pre-tRNA and tRNA but not on 12 nt trailer was confirmed by EMSA method 2 as described below (Fig 2A–C).

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