Decapping is preceded by 3' uridylation in a novel pathway of bulk mRNA turnover

Abstract

Both end structures of eukaryotic mRNAs, namely the 5' cap and 3' poly(A) tail, are necessary for transcript stability, and loss of either is sufficient to stimulate decay. mRNA turnover is classically thought to be initiated by deadenylation, as has been particularly well described in Saccharomyces cerevisiae. Here we describe two additional, parallel decay pathways in the fission yeast Schizosaccharomyces pombe. First, in fission yeast mRNA decapping is frequently independent of deadenylation. Second, Cid1-dependent uridylation of polyadenylated mRNAs, such as act1, hcn1 and urg1, seems to stimulate decapping as part of a novel mRNA turnover pathway. Accordingly, urg1 mRNA is stabilized in cid1Delta cells. Uridylation and deadenylation act redundantly to stimulate decapping, and our data suggest that uridylation-dependent decapping is mediated by the Lsm1-7 complex. As human cells contain Cid1 orthologs, uridylation may form the basis of a widespread, conserved mechanism of mRNA decay.

Figure 1. Decapping of mRNA can be independent of deadenylation

(a) Overview of the cRACE procedures used to capture ends of decapped transcripts (gray) and mature transcripts (white). For details see Methods. (b, c) The 5′ (b) and 3′ (c) ends of various types of act1 cRACE sequences are plotted as the distance (in nt, as indicated on the horizontal axis) from the start codon. The open reading frame (ORF) is marked with a line. (d) Poly(A) tail lengths of decapped [black; 40 sequences] and capped [white; 20 sequences] act1 cRACE sequences were binned into groups of ten nt. Tail lengths were then plotted as the percentage of adenylated species. (e) Box-and-whisker plots of poly(A) tail lengths found on six different decapped transcripts, act1, adh1, gar2, hcn1, pof9 and urg1 (n=40, 16, 11, 12, 8 and 39 respectively). This plot depicts the quartiles of poly(A) tail length with the whiskers representing the range of each data set, the boxplot demarcating the second and third quartiles, which are separated by the median.

Figure 2. Decapped mRNAs are often uridylated

(a) The percentage of decapped, adenylated cRACE products that contain terminal uridyl residues is shown for act1, adh1, gar2, hcn1, pof9 and urg1 (n= 10/40, 4/16, 5/11, 3/12, 2/8 and 7/39 respectively). (b) The poly(A) tail lengths of non-uridylated [black] and uridylated [white] decapped urg1 RNAs were binned into groups of ten nt. Tail lengths were then plotted as the percentage of adenylated species. (c) The poly(A) tail lengths of all non-uridylated [black; 31 sequences] and uridylated [white; 95 sequences] decapped transcripts are compared. For each transcript, each tail length was normalized to the median of non-uridylated tail length to correct for inter-transcript poly(A) tail length variability. These normalized lengths were then binned into groups and plotted as the percentage of adenylated species. (d) As in (b), the poly(A) tail lengths of non-uridylated [black] and uridylated [white] decapped act1 (b) were binned into groups of ten nt. Tail lengths were then plotted as the percentage of adenylated species.

Figure 3. mRNA uridylation is Cid1-dependent and impairment of decapping increases uridylation on decapped messages

(a) The percentage of adenylated, decapped act1 sequences that contain [black] or lack [white] terminal uridyl residues is plotted for sequences isolated from WT, cid1∆ and dcp1-ts cells (n=40, 29 and 20 respectively). (b, c) Poly(A) tail lengths, binned into groups of ten nt, of decapped (b) and capped (c) act1 sequences isolated from WT [black; n=40 and 20 respectively] and cid1∆ [white; n=29 and 36 respectively] cells are compared. (d, e) Poly(A) tail lengths, binned into groups of ten, of decapped (d) and capped (e) act1 sequences isolated from WT [black; n=40 and 20 respectively] and dcp1-ts [white; n=29 and 18 respectively] cells are compared.

Figure 4. Uridylation precedes decapping

(a, b) HSC-RACE products of total act1 transcripts isolated from WT cells (a) and dcp1-ts cells (b). The final four codons and stop codon [marked by *] are shown as well as the 3′ UTR. White boxes denote sequences with pure poly(A) tails; grey boxes denote poly(A) tails with internal non-adenyl residues; black boxes denote poly(A) tails with terminal uridyl residue(s). (c) Poly(A) tail lengths, binned into groups of ten nt, of HSC-RACE act1 species from WT [white bars] and dcp1-ts [black bars] cells. (d) Percentage of adenylated HSC-RACE act1 products that contain [black] or lack [white] terminal uridyl residues. (e) The percentage of capped hcn1 transcripts that contain [black] or lack [white] terminal uridyl residues is compared for RNA isolated from WT and dcp1-ts cells (n=18 and 14 respectively).

Figure 5. urg1 mRNA is more stable in cells lacking Cid1

(a-c) Northern blots of urg1 transcript remaining at various time-points after uracil washout (top panel) in WT (a), cid1∆ (b) and ccr4∆ cells (c). urg1 levels were normalized to pik1 mRNA (bottom panel). The percentage of urg1 remaining at each time point (shown below the bottom panel) was calculated by comparison to the normalized amount at 0 minutes. (d) The percentage of urg1 mRNA remaining after uracil wash-out is shown for three strains, WT (open circles), ccr4∆ (black squares) and cid1∆ (open triangles). At least three independent replicates were performed; error bars represent SEM. (e) The half-life of urg1 mRNA in different strains is shown. At least two independent replicates were performed for each strain. * denotes p<0.01; ** denotes p<0.001. Error bars denote standard deviation.

Figure 6. Deadenylation and uridylation function as redundant pathways in mRNA decay

(a) The percentage of decapped, adenylated urg1 sequences that contain [black] or lack [white] terminal uridyl residues is compared for RNA isolated from WT, ccr4∆, cid1∆ cells, pan2∆ cells and pan3∆ cells (n=39, 19, 19, 27 and 20 respectively). (b-d) Poly(A) tail lengths, binned into groups of ten nt, of decapped urg1 mRNAs isolated from (b) cid1∆ cells [white], (c) pan∆ cells [white] and (d) ccr4∆ cells [white] compared to those products from wild-type cells [black].

Figure 7. Uridylation-mediated decapping requires Lsm1

(a, b) Poly(A) tail lengths, binned into groups of ten nt, of capped (a) and decapped (b) act1 sequences isolated from WT [black; n=20 and 40 respectively] and lsm1∆ [white; n=19 and 22 respectively] cells are compared. (c) The percentage of decapped, adenylated act1 sequences that contain [black] or lack [white] terminal uridyl residues is compared for RNA isolated from WT and lsm1∆ cells.

Figure 8. A comparison of decay pathways for bulk mRNA in S. cerevisiae and S. pombe

See text for details.