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. 2006 Jan;12(1):26-32.
doi: 10.1261/rna.2207206.

Contributions of Trf4p- and Trf5p-dependent polyadenylation to the processing and degradative functions of the yeast nuclear exosome

Defne E Egecioglu  1 , Anthony K Henras, Guillaume F Chanfreau
Affiliations

Contributions of Trf4p- and Trf5p-dependent polyadenylation to the processing and degradative functions of the yeast nuclear exosome

Defne E Egecioglu et al. RNA. 2006 Jan.

Abstract

The nuclear exosome is involved in a large number of RNA processing and surveillance pathways. RNase III cleavage intermediates destined to be 3'-processed or degraded can be detected when the Rrp6p subunit of the nuclear exosome is absent. Here we show that these processing and degradation intermediates are polyadenylated, and that their polyadenylation is dependent on the activity of Trf4p and Trf5p, two variant poly(A) polymerases. Polyadenylation of cleavage intermediates was inhibited when Trf4p was absent, and reduced to various extents in the absence of Trf5p, suggesting that these two poly(A) polymerases play functionally distinct roles in the polyadenylation of these RNA species. Finally, in the absence of Trf4p, we observed 3'-extended forms of the U4 snRNA that are similar to those observed in the absence of Rrp6p. These results suggest that polyadenylation of RNA processing intermediates plays a functional role in RNA processing pathways and is not limited to RNA surveillance functions.

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Figures

FIGURE 1.
FIGURE 1.
Analysis of the in vivo polyadenylation status of Rnt1p cleavage products and primary precursors. (A) Schematic representation of the primary precursors of the U4 snRNA, the U3 and snR40 snoRNAs, and the RPL18A and RPS22B mRNAs. Boxes represent mature or exonic sequences. Arrows represent the Rnt1p cleavage sites. Black lines with asterisks represent the location of the oligonucleotide probes used. (B) 3′-Processing intermediates and unprocessed precursors of the U3 snoRNA and of the U4 snRNA are polyadenylated. Total RNAs (T) or polyadenylated RNAs selected by oligo-dT affinity (P) extracted from the indicated strains were loaded on acrylamide gels, transferred to nylon membranes, and probed with oligonucleotides hybridizing to the mature sequences of the U3 snoRNA or U4 snRNA. Species with arrowheads represent the species that result from Rnt1p cleavage. The Arginine tRNA tR(UCU) was used as a negative control to test for contamination of purified polyadenylated RNAs by total RNAs. (C) The upstream degradation intermediate of the snR40 box C/D snoRNA is polyadenylated. Legends as in B. The probe used hybridizes against the 5′-extension of snR40 (see A); therefore, the mature snR40 snoRNA is not detected. (D) Degradation intermediates of the RPL18A and RPS22B unspliced pre-mRNAs are polyadenylated. Legends as in B. Probes used hybridize against the 5′-exon sequences of RPL18A or RPS22B. The same cleaved intermediate was detected by using an intronic oligonucleotide probe in the case of RPS22B (data not shown). For RPS22B, the unspliced precursor is not included in the gel.
FIGURE 2.
FIGURE 2.
Polyadenylation of Rnt1p cleavage intermediates requires Trf4p. (A) Analysis of the polyadenylation status of the U3 and U4 cleavage intermediates in trf4Δ-derived strains. The membrane was also hybridized with the RPL18A probe to test for equal loading in the poly(A)+RNA lanes. (B) 3′-Extended forms of the U4 snRNA accumulate in the absence of Trf4p. A membrane with total RNAs extracted from wild-type, trf4Δ, or rrp6Δ strains was hybridized with the U4 probe. The brace indicates the 3′-extended forms that accumulate in the absence of Trf4p or Rrp6p. (C) Analysis of the polyadenylation status of the RPL18A and snR40 cleavage intermediates in trf4Δ-derived strains. Legends as in Figure 1 ▶.
FIGURE 3.
FIGURE 3.
Polyadenylation of Rnt1p cleavage intermediates is influenced by Trf5p. (A) Analysis of the polyadenylation status of the U3 and U4 cleavage intermediates in trf5Δ-derived strains. The membrane was also hybridized with the RPL18A probe to test for equal loading in the poly(A)+RNA lanes. (B) Analysis of the polyadenylation status of the RPL18A and snR40 cleavage intermediates in trf5Δ-derived strains. Legends as in Figure 1 ▶. (C) The reduction of polyadenylated cleavage intermediates observed in the rrp6Δ trf5Δ double deletion strain for the U3 and U4 RNAs is reproducible. “A” and “B” represent RNAs extracted from two independent clones carrying the rrp6Δ trf5Δ double deletion; “1” and “2” represent two independent RNA preparations.
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