CUCU modification of mRNA promotes decapping and transcript degradation in Aspergillus nidulans

Abstract

In eukaryotes, mRNA decay is generally initiated by the shortening of the poly(A) tail mediated by the major deadenylase complex Ccr4-Caf1-Not. The deadenylated transcript is then rapidly degraded, primarily via the decapping-dependent pathway. Here we report that in Aspergillus nidulans both the Caf1 and Ccr4 orthologues are functionally distinct deadenylases in vivo: Caf1 is required for the regulated degradation of specific transcripts, and Ccr4 is responsible for basal degradation. Intriguingly disruption of the Ccr4-Caf1-Not complex leads to deadenylation-independent decapping. Additionally, decapping is correlated with a novel transcript modification, addition of a CUCU sequence. A member of the nucleotidyltransferase superfamily, CutA, is required for this modification, and its disruption leads to a reduced rate of decapping and subsequent transcript degradation. We propose that 3' modification of adenylated mRNA, which is likely to represent a common eukaryotic process, primes the transcript for decapping and efficient degradation.

FIG. 1.

Northern analysis of areA transcript degradation in deadenylase mutants. (A) Northern blot analysis of areA (encoding a global transcription factor mediating nitrogen regulation), niaD (encoding nitrate reductase), and meaA (encoding a high-affinity ammonium transporter) was conducted over a 30-min time course, after transcription was inhibited, to monitor degradation rates under conditions of nitrogen starvation (−N) or nitrogen sufficiency (Gln). The wild type (WT) is compared with strains with ccr4 or caf1 deleted, as indicated. 18S rRNA was used as a loading control. Multiple Northern blots (≥3) were quantified, and the data are represented graphically (±standard deviations [SD]), with the y axis being a logarithmic plot of the percentage of transcript remaining (□, −N; •, Gln). (B) Extrapolated half-lives, derived by regression analysis. Between the two growth regimens there was a significant difference (t test, P < 0.05) in half-life for all three transcripts in the wild-type and Δccr4 strains. Conversely, in the Δcaf1 strain no significant difference was observed (areA, P = 0.216; niaD, P = 0.572; meaA, P = 0.865).

FIG. 2.

RNase H analysis of areA poly(A) tail length in deadenylase mutants. RNase H Northern blot analysis was used to monitor the rate of the deadenylation over a 30-min time course for the areA transcript in the presence (Gln) or absence (−N) of glutamine with or without cycloheximide (CX) as indicated. Transcription was inhibited prior to the time course being initiated. The wild type (WT) is compared with strains with ccr4 or caf1 deleted, as indicated. A separate experiment comparing time zero samples for all three strains on the same gel confirmed that there is no major difference with respect to the maximal poly(A) tail length observed (data not shown). 18S rRNA is used as a loading control. The T₀ sample was treated with RNase H and oligo(dT) and utilized as a size marker for deadenylated transcripts (A₀). As noted previously, 3′ degradation proceeds beyond the poly(A) tail after cycloheximide treatment in the wild type (47); this is also the case for the Δcaf4 strain but not the Δcaf1 strain.

FIG. 3.

Poly(A) tail profile. (A) The poly(A) tail length distribution for bulk mRNA was assayed by 3′ end labeling of total RNA. RNA samples were equivalent to the T₀ and T₃₀ Gln plus cycloheximide samples used in Fig. 2. The samples assayed are from the Δccr4 strain at T₀ (lane 1) and T₃₀ (lane 2), from the wild-type (WT) strain at T₀ (lane 3) and T₃₀ (lane 4), and from the Δcaf1 strain at T₀ (lane 5) and T₃₀ (lane 6). (B) The profile for each T₀ sample was obtained using a phosphorimager, and the data from triplicate experiments were combined prior to normalization. The standard errors are indicated. A DNA sequencing run was included on the gels to provide a size marker. As with RNase H analysis, distinct profiles were obtained; in particular, the Δcaf1 strain shows a relatively small proportion of transcripts with very short poly(A) tails.

FIG. 4.

cRT PCR analysis of gdhA mRNA. (A) Poly(A) tail length was determined by cRT PCR and sequencing of RNA samples derived from the wild-type, Δcaf1, ΔcutA, and Δccr4 strains, with (+) or without (−) pretreatment with TAP to remove the 5′ cap structure. The distribution of the poly(A) tail lengths is displayed using a box plot, where the top and bottom of the box represent limits of the upper and lower quartiles, with the median being indicated by the horizontal line which lies within the box. The whiskers show the highest and lowest reading within 1.5 times the interquartile range. The outliers are indicated (□). Projected onto this is the distribution of clones that include CUCU-derived modifications (○). The sequences of specific modifications identified and their respective poly(A) tail lengths are shown. These data are derived from three separate experiments. The total numbers of transcripts analyzed are also given. Based on semiquantitative PCR, the wild-type TAP-untreated sample had <2% uncapped transcripts, compared to the treated sample (see Fig. S3 in the supplemental material). (B) Distribution of poly(A) tail lengths in the wild type (WT) and ΔcutA strains without pretreatment with TAP. The x axis shows those with a tail in A₈ groups.

FIG. 5.

Northern analysis of areA transcript degradation in the ΔcutA mutant. (A) Northern blot analyses of areA, gdhA, and meaA mRNA under conditions of nitrogen starvation (−N) or nitrogen sufficiency (Gln) were conducted as for Fig. 1. The wild type (WT) is compared with the strain with cutA deleted, as indicated. 18S rRNA was used as a loading control. Multiple Northern blots (≥3) were quantified for the wild type (dashed lines) incubated in the absence of nitrogen (○) or in the presence of Gln (•) or the ΔcutA strain (solid lines) incubated in the absence of nitrogen (□) or in the presence of Gln (▪), and the data were represented graphically (±SD), with the y axis being a logarithmic plot of the percentage of transcript remaining. (B) The extrapolated half-lives for areA, gdhA, and meaA transcripts, under both nitrogen regimens, are given to facilitate comparison of the three strains.