Competition between the Rex1 exonuclease and the La protein affects both Trf4p-mediated RNA quality control and pre-tRNA maturation

Abstract

Although nascent noncoding RNAs can undergo maturation to functional RNAs or degradation by quality control pathways, the events that influence the choice of pathway are not understood. We report that the targeting of pre-tRNAs and certain other noncoding RNAs for decay by the TRAMP pathway is strongly influenced by competition between the La protein and the Rex1 exonuclease for access to their 3' ends. The La protein binds the 3' ends of many nascent noncoding RNAs, protecting them from exonucleases. We demonstrate that unspliced, end-matured, partially aminoacylated pre-tRNAs accumulate in yeast lacking the TRAMP subunit Trf4p, indicating that these pre-tRNAs normally undergo decay. By comparing RNA extracted from wild-type and mutant yeast strains, we show that Rex1p is the major exonuclease involved in pre-tRNA trailer trimming and may also function in nuclear CCA turnover. As the accumulation of end-matured pre-tRNAs in trf4Delta cells requires Rex1p, these pre-tRNAs are formed by exonucleolytic trimming. Accumulation of truncated forms of 5S rRNA and SRP RNA in trf4Delta cells also requires Rex1p. Overexpression of the La protein Lhp1p reduces both exonucleolytic pre-tRNA trimming in wild-type cells and the accumulation of defective RNAs in trf4Delta cells. Our experiments reveal that one consequence of Rex1p-dependent 3' trimming is the generation of aberrant RNAs that are targeted for decay by TRAMP.

FIGURE 1.

Several noncoding RNAs accumulate in trf4Δ cells. (A) RNA extracted from the indicated strains was subjected to Northern blotting to detect precursor and mature forms of tRNA^Ser _CGA, tRNA^Arg _CCG, U4 and U6 snRNAs, SRP RNA, U3 snoRNA, and 5S rRNA. (Arrow) A truncated form of SRP RNA that is present in trf4Δ cells and more abundant in trf4Δ rrp6Δ cells. To visualize pre-U3 snoRNA, a longer exposure was obtained of the U3 panel. (Asterisk) A pre-U3 RNA degradation product. All strains were grown at 25°C. (B) To quantitate the differences in levels of the end-matured unspliced pre-tRNA^Ser _CGA and mature tRNA^Ser _CGA between wild-type, trf4Δ, lhp1Δ, and trf4Δlhp1Δ cells, the intensity of each band was determined and the values normalized relative to the amount of the 5S rRNA. Data from three independent experiments are graphed. For each experiment, the intensity of the wild-type band was set to 1.0. (C) Fivefold serial dilutions of the indicated strains were spotted onto YPD agar and grown at 25°C, 30°C, and 37°C. Because growth differences between rrp6Δ trf4Δ and rrp6Δ trf4Δ lhp1Δ cells at 37°C were not visible after 2 d, the plates were also photographed after 4 d.

FIGURE 2.

Many end-matured unspliced pre-tRNAs accumulate in trf4Δ cells. (A) RNA extracted from wild-type, trf4Δ, lhp1Δ, and trf4Δ lhp1Δ cells were subjected to Northern blotting to detect the indicated intron-containing pre-tRNAs and mature tRNAs. As a loading control, blots were reprobed to detect 5S rRNA. (B) To better resolve small differences in mobility, the RNAs shown in A were fractionated in a high-resolution sequencing gel and subjected to Northern blotting to detect precursor and mature tRNA^Lys _UUU. (C) RNAs extracted from the indicated strains were subjected to Northern blotting to detect precursor and mature forms of tRNA^Ser _CGA. Blots were reprobed to detect 5S rRNA. (D) Wild-type, lhp1Δ, trf4Δ, and trf4Δ rrp6Δ extracts were subjected to immunoprecipitation with anti-Lhp1p antibodies. RNAs in immunoprecipitates, supernatants, and an equivalent amount of the extracts were subjected to Northern analysis to detect precursor and mature tRNA^Ser _CGA.

FIGURE 3.

Rex1p functions in pre-tRNA maturation. (A,B) RNAs from the indicated strains were subjected to Northern blotting to detect precursor and mature forms of (top) tRNA^Ser _CGA, (middle) tRNA^Tyr _GUA, and (bottom) tRNA^Lys _UUU. As a loading control, blots were reprobed to detect 5S rRNA. In rex1Δ cells, the 5S rRNAs are ∼3 nt longer at the 3′ end (van Hoof et al. 2000). The dots in panel A designate forms of pre-tRNA^Tyr _GUA and pre-tRNA^Lys _UUU, containing a mature 5′ end and a 3′ trailer, that increase in abundance when Rex1p is deleted.

FIGURE 4.

Rrp6 and Rex1p participate in pre-tRNA maturation in lhp1Δ cells. (A) Fivefold serial dilutions of the indicated strains were spotted on YPD agar and grown for 3 d at 25°C. (B) RNA from the indicated strains were subjected to Northern blotting to detect precursor and mature forms of tRNA^Ser _CGA and tRNA^Tyr _GUA. As a loading control, blots were reprobed to detect 5S rRNA.

FIGURE 5.

Rex1p is required for the accumulation of aberrant RNAs in trf4Δ cells. (A) RNA from the indicated strains was fractionated in 8% polyacrylamide, 8.3 M urea gels and subjected to Northern blotting to detect precursor and mature forms of (top) tRNA^Ser _CGA, (middle) tRNA^Tyr _GUA, and (bottom) tRNA^Lys _UUU. The dots designate forms of pre-tRNA^Tyr _GUA and pre-tRNA^Lys _UUU, containing mature 5′ ends and 3′ trailers, that increase in abundance in rex1Δ cells. As a loading control, the blot was reprobed to detect 5S rRNA. (B) RNA from wild-type and trf4Δ cells carrying LHP1 on (lanes 2,4) a low-copy plasmid or (lanes 1,3) the plasmid alone was subjected to Northern blotting to detect the indicated RNAs. The pre-tRNA^Tyr _GUA containing a mature 5′ end and a 3′ trailer that is more abundant when LHP1 is overexpressed in (lane 2) wild-type cells is indicated by a dot. (C) RNA from the indicated strains was fractionated in 5% gels and subjected to Northern blotting to detect full-length and truncated SRP RNA and 5S rRNA. To detect the truncated 5S rRNA, a longer exposure was obtained of the 5S panel (bottom). The truncated RNAs are indicated with arrows.

FIGURE 6.

Pre-tRNAs that accumulate in trf4Δ and rex1Δ cells contain CCA. (A) RNA from wild-type, lhp1Δ, rex1Δ, and rex1Δ lhp1Δ cells was fractionated in a high-resolution gel and subjected to Northern blotting to detect pre-tRNAs. The four end-matured forms of pre-tRNA^Tyr _GUA in rex1Δ cells are indicated by dots. (B) RNA from the indicated strains was subjected to Northern analyses to detect precursor and mature tRNA^Tyr _GUA. After all strains were grown at 25°C, the rna1-1 and cca1-1 strains were shifted for 2 h to 37°C. (C) RNA from the indicated strains was incubated with oligonucleotides complementary (lanes 2,5,8,11) to the pre-tRNA containing a partly or fully cleaved trailer or (lanes 3,6,9,12) to the CC- and CCA-containing pre-tRNA and extended with T7 DNA polymerase. (Lanes 1,4,7,10) No oligonucleotide was added. Following gel fractionation, Northern analyses were performed to detect pre-tRNA_GUA ^Tyr. Extended pre-tRNAs that contain a fully or partly cleaved trailer are indicated by circles, while extended pre-tRNAs containing CC- or CCA are indicated by diamonds. Similar results were obtained when the blot was reprobed to detect pre-tRNA^Ser _CGA (data not shown). (D) RNA from wild-type and trf4Δ cells was extracted under acidic conditions, fractionated in acidic acrylamide gels, and subjected to Northern blotting to detect precursor and mature forms of tRNA^Tyr _GUA.

FIGURE 7.

Model for pre-tRNA 3′-end maturation. (A) In wild-type cells, Lhp1p protects the 3′ ends of many nascent pre-tRNAs from exonucleases. Thus, the first cleavage is by RNase P, resulting in an Lhp1p-bound pre-tRNA with a mature 5′ end and a 3′ extension. The trailer is then removed by an endonuclease such as RNase Z. For other pre-tRNAs, Lhp1p either does not bind or dissociates, allowing Rex1p to trim the trailer up to a helix formed by base-pairing between the leader and trailer (Lee et al. 1997; Yoo and Wolin 1997). Following removal of the leader by RNase P, Rex1p and other exonucleases mature the 3′ end. It is also possible that the 3′ endonuclease removes the remaining trailer sequences. Following CCA addition, Rex1p can remove the terminal adenosine. (B) In lhp1Δ cells, Rex1p, Rrp6p, and other exonucleases contribute to the removal of the trailer. As in wild-type cells, the 3′ endonuclease may also contribute to trailer removal. Following CCA addition, Rex1p participates in end turnover.