pubmed.ncbi.nlm.nih.gov

Rrp6p controls mRNA poly(A) tail length and its decoration with poly(A) binding proteins - PubMed

  • ️Sun Jan 01 2012

Rrp6p controls mRNA poly(A) tail length and its decoration with poly(A) binding proteins

Manfred Schmid et al. Mol Cell. 2012.

Abstract

Poly(A) (pA) tail binding proteins (PABPs) control mRNA polyadenylation, stability, and translation. In a purified system, S. cerevisiae PABPs, Pab1p and Nab2p, are individually sufficient to provide normal pA tail length. However, it is unknown how this occurs in more complex environments. Here we find that the nuclear exosome subunit Rrp6p counteracts the in vitro and in vivo extension of mature pA tails by the noncanonical pA polymerase Trf4p. Moreover, PABP loading onto nascent pA tails is controlled by Rrp6p; while Pab1p is the major PABP, Nab2p only associates in the absence of Rrp6p. This is because Rrp6p can interact with Nab2p and displace it from pA tails, potentially leading to RNA turnover, as evidenced for certain pre-mRNAs. We suggest that a nuclear mRNP surveillance step involves targeting of Rrp6p by Nab2p-bound pA-tailed RNPs and that pre-mRNA abundance is regulated at this level.

Copyright © 2012 Elsevier Inc. All rights reserved.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Rrp6p is required for normal pA tail length

(A) The sequence of the CYC1s substrate used in this study is indicated by a double-arrowed line above the relevant CYC1 3′UTR sequence. Numbering is relative to the CYC1 ORF stop codon. Polyadenylation efficiency- (left) and positioning- (right), elements are underlined. Major cleavage sites are marked by vertical arrows. (B) CYC1s in vitro polyadenylation time course in wt, pap1-1 and trf4Δ extracts. Positions of precursor (CYC1s), short and mature polyadenylated species are indicated to the right and pA tail lengths estimated from the migration of DNA size markers are shown to the left of the gel. (C) Phosphorimager lane scans from wt reactions shown in (B) carried out for 15 min (black), 30 min (dark gray) or 60 min (light gray). The position of the precursor (42 nt) is indicated by an arrow. Areas covering short (5–20 nt) and mature (60–80 nt) pA tail regions are marked by double arrows and shaded. (D) CYC1s in vitro polyadenylation time course in wt, rrp6Δ, pan2Δ and pan3Δ extracts. Image labeling as in (B). ‘Hyperadenylated’ denotes excess adenylation over the normal mature (wt) length. (E) Lane scans of 15 min (light gray) and 90 min (dark gray) time points of wt (top), rrp6Δ (middle) and pan2Δ (bottom) reactions from (D). Image labeling as in (C). The region of hyperadenylation is indicated by an asterisk.

Figure 2
Figure 2. In the absence of Rrp6p, Trf4p extends Pap1p-produced tails

(A) CYC1s in vitro polyadenylation time course in wt, rrp6Δ, trf4Δ and rrp6Δ/trf4Δ extracts. Image labeling as in Figure 1D. A minor hyperadenylation phenotype is observed in trf4Δ extracts, which is likely due to lower levels of Pab1p in these extracts (data not shown), leading to decreased Pan2p/Pan3p deadenylation. (B) Lane scans of the 15 min (top) and 90 min (bottom) reactions from wt (black), rrp6Δ (light gray), trf4Δ (medium gray) or rrp6Δ/trf4Δ (dark gray) extracts from (A). Image labeling as in Figure 1C and 1E. (C) CYC1s in vitro polyadenylation reactions performed for 30 min in non-depleted (‘total’, lanes 6 and 8) or Trf4p-TAP depleted (‘dep’ lanes 7 and 9) TRF4-TAP and rrp6Δ/TRF4-TAP extracts. ‘Total’ and ‘dep’ versions of untagged extracts (lanes 2–5) were included as controls. Trf4p-TAP was depleted using magnetic beads coated with rabbit IgG (Figure S1A). Image labeling as in (A). (D) Lane scans of reactions in (C) labeled as in (B). Total (dark colors) and depleted (light colors) lanes are overlaid in each panel. (E) In vivo pA tail length measurements of total RNA purified from wt, rrp6Δ, trf4Δ or rrp6Δ/trf4Δ cells grown at 25°C, 30°C or temperature-shifted to 37°C for 60 min as indicated. pA tail lengths estimated from the migration of DNA size markers are shown to the left. See also Figure S1.

Figure 3
Figure 3. Efficient Nab2p incorporation into pA tail RNP only occurs in the absence of Rrp6p

(A) CYC1s in vitro polyadenylation reactions carried out for 30 min in wt or rrp6Δ extracts were terminated by the addition of excess dATP and subjected to IP by magnetic beads containing no (mock), α-Pab1p or α-Nab2p antibody. RNA was collected from input, unbound and bead-bound fractions and resolved by denaturing PAGE. Quantification of relative IP efficiencies of mature and hyperadenylated RNA species (see Experimental Procedures) are shown below the images labeled as in Figure 2A. The first lane (not numbered) was loaded with an equivalent amount of CYC1s precursor. (B) Same as in (A) but using trf4Δ and rrp6Δ/trf4Δ extracts for polyadenylation reactions. (C) Pab1p and Nab2p association with Cbp20p-bound RNPs in vivo. Cbp20p-TAP was purified using magnetic beads coated with rabbit IgG from wt or rrp6Δ cells and eluates were examined by western blotting analysis for their contents of Cbp20p-TAP, Cbp80p, Pab1p and Nab2p. ‘No TAP’ denotes a negative control strain carrying un-tagged Cbp20p. 0.5% of the total input (lanes 1–3) was loaded next to IP reactions carried out without (lanes 4–6) or with (lanes 7–9) addition of an RNase cocktail. Longer exposures of Cbp20p-TAP and Cbp80p blots are shown to reveal the presence of these proteins in input lanes. See also Figure S2.

Figure 4
Figure 4. Rrp6p can displace pre-loaded Nab2p from pA tails

(A) Schematic outline of the experiment analyzed in (B). See text for details. (B) CYC1s polyadenylation reactions were carried out for 30 min in Pab1p-TAP or rrp6Δ/Pab1p-TAP extracts and then incubated with rabbit IgG Dynabeads for an additional 15 min to capture Pab1p-TAP (‘total’ lanes 2 and 6). Pab1p-TAP containing RNPs were then isolated (‘bound’, lanes 3 and 7) and aliquots of bead-bound material were incubated for 30 min in a chase reaction with either wt (lanes 4 and 8) or rrp6Δ (lanes 5 and 9) extracts. RNA isolated from the different steps was visualized by denaturing PAGE (‘RNA’) with image labeling as in Figure 1B, whereas the content of Pab1p-TAP and Nab2p in the samples was analyzed by western blotting analysis (‘protein’). The asterisk at the anti-Pab1p western image indicates a putative degradation fragment of Pab1p-TAP. Bottom: Scans of the indicated lanes as in Figure 1C and 1E. (C) Pull-down of Nab2p interaction partners. GST-Nab2p or GST (negative control) was immobilized on Sepharose columns and incubated with wt, Δrrp6 or Δtrf4 cell extract as indicated. Columns were washed and eluates were probed by western blotting analysis for their presence of Rrp6p, Trf4p and Pab1p (top), and by Coomassie staining to control for equal elution of GST-Nab2p and GST between experiments (bottom). Similar fractions of input material (lanes 1–3), and eluate (lanes 4–9) were loaded. The GST-Nab2p IP of wt extract was carried out in parallel with (lane 10) or without (lane 11) addition of the nuclease Benzonase. Note that the RNA-mediated binding of Pab1p to GST-Nab2p is lost upon Benzonase addition, whereas the interactions of Rrp6p and Trf4p are not. An unspecific band migrating slightly lower than Trf4p in the Trf4p panel is marked with an asterisk. See also Figure S3.

Figure 5
Figure 5. Nab2p is not required for normal pA tail biogenesis

(A) CYC1s in vitro polyadenylation reactions carried out for 30 min in wt or rrp6Δ extracts before (total) or after (dep) no TAP-, Pab1p-TAP- or Nab2p-TAP-depletion as indicated. Pab1p-TAP and Nab2p-TAP proteins were depleted using rabbit IgG antibody. Image labeling as in Figure 1B. (B) CYC1s in vitro polyadenylation reactions carried out for 10 or 30 min in wt extracts supplemented with buffer (lanes 2 and 3), 0.5-fold (lanes 4 and 5) or 2-fold (lanes 6 and 7) excess recombinant Pab1; or 2-fold (lanes 8 and 9) or 8-fold (lanes 10 and 11) excess recombinant Nab2 as indicated. See also Figure S4.

Figure 6
Figure 6. Nab2p and Rrp6p regulate pre-mRNA levels

(A) and (B) Boxplots (see Supplemental Experimental Procedures) of global expression changes of indicated transcript classes identified by tiling microarray analysis. Individual plots depict log2-fold expression changes comparing total RNA from THC-NAB2 cells depleted for 7h (black) or 12h (dark gray) relative to non-depleted (0h) samples, as well as total RNA from rrp6Δ relative to wt (light gray) cells. Note that Nab2p-depletion data cDNA was obtained from total RNA, whereas pA-enriched RNA was used for wt and rrp6Δ samples. Hence, signal values for most transcripts are lower for THC-NAB2 than for rrp6Δ samples. ‘All transcripts’ include exons and introns of protein-encoding transcripts as well as non-coding transcripts. Panel ‘ORF-T’ (top, middle panel) includes only exons of protein-encoding transcripts (ORF-Ts). RP: Ribosomal protein. (C) Screenshots of tiling array tracks for five RP genes, displaying normalized probe signal intensities of individual probes (gray-scaled as shown on the right) of three independent biological replicates of THC-NAB2 12h, 7h and 0h samples as well as duplicates of rrp6Δ and wt samples (see text for details). Position of introns and transcription directions are indicated by dashed boxes and arrows, respectively. Vertical lines indicate refer to transcript boundaries. Calculated log2-fold changes for displayed exonic and intronic RNA are shown at the bottom. (D) Validation of results displayed in (C) using oligo(dT)-directed qPCR with amplicons spanning exon-intron junctions (‘pre-mRNA’, left panels) or amplicons entirely within exon2 (‘mRNA+pre-mRNA’, right panels). Expression values depicted are normalized to the intron-less mRNA PMA1 and to the 0h time point for the THC-NAB2 strain (top panel) or to the wt control in the bottom panel. Error bars are from triplicate PCRs of THC-NAB2 culture 1 (similar results were confirmed for culture 2 and 3, data not shown) and for untreated wt and rrp6Δ cultures. See also Figure S5 and Table S1.

Figure 7
Figure 7. Models of functional significance of Nab2p-Rrp6p targeting

Nuclear pA-tails are either loaded with Pab1p (A), Nab2p (B) or a mixture of the two (C). (A) In wt cells, the majority of mRNA pA tails are Pab1p-bound and destined for nuclear export and cytoplasmic translation. Pab1p recruits the PAN deadenylase complex, but the location in which this interaction is functionally significant is presently unclear (indicated by question marks). The influence of Pab1p and Nab2p on the activity of the major deadenylase activity from the Ccr4/NOT complex (not shown) is not known. (B) Nab2p-bound tails may escape nuclear targeting by Rrp6p by rapid export. In the cytoplasm, Nab2p is exchanged for Pab1p. If export is delayed, Rrp6p is recruited for complete mRNA destruction. This pathway is involved in quality control and gene expression regulation. (C) Pab1p-decorated nuclear pA tails harboring Nab2p at their 3′ends can target Rrp6p for trimming and pA tail length control. Symbols: Pab1p (oval), Nab2p (hexagon), PAN complex (light pacman symbol), Rrp6p (dark pacman symbol). See text for further details.

Similar articles

Cited by

References

    1. Amrani N, Minet M, Le Gouar M, Lacroute F, Wyers F. Yeast Pab1 interacts with Rna15 and participates in the control of the poly(A) tail length in vitro. Mol Cell Biol. 1997;17:3694–3701. - PMC - PubMed
    1. Assenholt J, Mouaikel J, Andersen KR, Brodersen DE, Libri D, Jensen TH. Exonucleolysis is required for nuclear mRNA quality control in yeast THO mutants. Rna. 2008;14:2305–2313. - PMC - PubMed
    1. Boeck R, Lapeyre B, Brown CE, Sachs AB. Capped mRNA degradation intermediates accumulate in the yeast spb8-2 mutant. Mol Cell Biol. 1998;18:5062–5072. - PMC - PubMed
    1. Bonnerot C, Boeck R, Lapeyre B. The two proteins Pat1p (Mrt1p) and Spb8p interact in vivo, are required for mRNA decay, and are functionally linked to Pab1p. Mol Cell Biol. 2000;20:5939–5946. - PMC - PubMed
    1. Bousquet-Antonelli C, Presutti C, Tollervey D. Identification of a regulated pathway for nuclear pre-mRNA turnover. Cell. 2000;102:765–775. - PubMed

Publication types

MeSH terms

Substances