A meiotic gene regulatory cascade driven by alternative fates for newly synthesized transcripts

Abstract

To determine the relative importance of transcriptional regulation versus RNA processing and turnover during the transition from proliferation to meiotic differentiation in the fission yeast Schizosaccharomyces pombe, we analyzed temporal profiles and effects of RNA surveillance factor mutants on expression of 32 meiotic genes. A comparison of nascent transcription with steady-state RNA accumulation reveals that the vast majority of these genes show a lag between maximal RNA synthesis and peak RNA accumulation. During meiosis, total RNA levels parallel 3' processing, which occurs in multiple, temporally distinct waves that peak from 3 to 6 h after meiotic induction. Most early genes and one middle gene, mei4, share a regulatory mechanism in which a specialized RNA surveillance factor targets newly synthesized transcripts for destruction. Mei4p, a member of the forkhead transcription factor family, in turn regulates a host of downstream genes. Remarkably, a spike in transcription is observed for less than one-third of the genes surveyed, and even these show evidence of RNA-level regulation. In aggregate, our findings lead us to propose that a regulatory cascade driven by changes in processing and stability of newly synthesized transcripts operates alongside the well-known transcriptional cascade as fission yeast cells enter meiosis.

FIGURE 1:

Comparison of nascent transcription with steady-state RNA accumulation for 32 meiotic and 2 vegetative genes. Row numbers correspond to those in Table 1. (A) Analyses of nascent transcription over a meiotic time course using TRO assays. Cells were harvested at the times indicated after meiotic induction and processed as described previously (McPheeters et al., 2009). Newly synthesized ³²P-labeled RNAs were detected by hybridization to filter-bound PCR fragments of ∼500 bp corresponding to sequences near the 3′ end of each ORF. The decreased background in the 5- to 8-h TRO assays reflects the lower overall incorporation of label during late meiosis (see Supplemental Figure S1). (B) Semiquantitative RT-PCR analyses of RNA accumulation over a meiotic time course. Total RNA was extracted at the times indicated after the temperature shift, and steady-state RNA levels were assayed by RT-PCR with the same primers used to generate PCR products for the TRO experiment in A; products were visualized negatively after ethidium bromide staining. Reactions were carried out for 21 cycles with three exceptions: for meu4 (row 13) and atg8 (row 23), the cycle number was reduced to 12 and 16, respectively, whereas the low abundance of the SPAC6F6.11c transcript (row 34) necessitated an increase to 28 cycles. Thick lines separate the early, middle, late, and vegetative genes (see Table 1), and thin lines separate every fifth middle meiotic gene for alignment purposes.

FIGURE 2:

Analyses of polyadenylation over a meiotic time course. Total RNA was extracted at the times indicated after the temperature shift and the presence of a poly(A) tail was assessed by RT-PCR using a 5′ primer near the 3′ end of the ORF and oligo(dT) as the 3′ primer, as described previously (McPheeters et al., 2009). D and P next to the row number indicate poly(A) sites distal and proximal to the ORF, respectively. The cycle number was 28 except in the case of meu4 (row 13), for which 12 cycles gave a strong signal, consistent with qRT-PCR data (Supplemental Figure S4C). For genes with two closely spaced cleavage/polyadenylation sites, both are displayed on the same gel slice; two separate slices are shown for more widely separated sites. Thick lines separate the early, middle, late, and vegetative genes (see Table 1). The position(s) of poly(A) tail addition (far right) were determined by sequencing of the indicated cDNAs and are expressed as distance (in nt) from the translational stop codon to the cleavage/polyadenylation site.

FIGURE 3:

Effect of including a nitrogen starvation step prior to shifting pat1–114 cells to the nonpermissive temperature. (Top) Accumulation of total RNA for representative early, middle, and vegetative transcripts using the same RT-PCR primers as in Figure 1B, which amplify the the 3′ ends of the coding regions. (Bottom) Accumulation of poly(A)⁺ RNA was analyzed for the same transcripts using RT-PCR with oligo(dT) as the 3′ primer, as in Figure 2.

FIGURE 4:

Effects of mutating factors implicated in meiotic and general nuclear RNA turnover on accumulation of meiotic transcripts. (A) Analysis of poly(A)⁺ RNA accumulation for mmi1 targets identified in the original study (Harigaya et al., 2006). The two temperature-sensitive mutants (ts3 and ts6) or an isogenic wild-type control (WT) were grown to early log phase at the permissive temperature (26ºC) and then shifted to the nonpermissive temperature (36ºC) for 3 h prior to RNA extraction. Polyadenylation assays were performed as in Figure 2. One of the 13 genes up-regulated in mmi1-ts mutants, SPCP20C8.03 (Harigaya et al., 2006), is not included as we were unable to map a polyadenylation site, consistent with the recent reclassification of the locus as a pseudogene. (B) Analysis of poly(A)⁺ RNA accumulation for a subset of the meiotic genes (see text under “Mutations in RNA surveillance factors…“) in the mmi1-ts, rrp6Δ, and cid14Δ strains. The mmi1 strains were processed as in A, while the deletion mutants were grown to early log phase at 30ºC (the standard growth temperature for fission yeast) before harvesting RNA. To assess the impact of mutations, we assigned a numerical value from 0 (undetectable) to 5 to the signal intensity in each lane; these values are shown at the right.

FIGURE 5:

Analyses of meu6 and atg8 splicing over a meiotic time course. Splicing was assayed by RT-PCR using primers complementary to the terminal exons (both genes contain two introns; Table 1). Signal-recognition particle (SRP) RNA was used as an internal loading control as in our previous study (McPheeters et al., 2009).

FIGURE 6:

Model for a meiotic gene regulatory cascade initiated by inactivation of a pathway that promotes turnover of meiotic transcripts produced in vegetatively growing cells. During proliferation (top), Mei2p is maintained in the inactive phosphorylated state by the Pat1p kinase (Iino and Yamamoto, 1985), and Mmi1p is available to target certain transcripts for destruction by the nuclear exosome, which contains Rrp6p. Upon entry into meiosis (below dotted line), Mmi1p is sequestered in a nuclear dot via association with Mei2p and the noncoding meiRNA (Harigaya et al., 2006), thereby allowing its substrate RNAs to accumulate. The only middle meiotic transcript known to be a direct target of the mmi1 pathway is mei4, which encodes a transcription factor that in turn regulates a large number of target genes (bottom). Some of these transcripts, highlighted in bold in both halves of the figure, also display enhanced accumulation in one or more surveillance mutants (Figure 4B). For most of the mei4 target genes, there is no discernible peak in transcription (Figure 1A; middle right); these genes include mei4 itself, which is subject to autoregulation (Abe and Shimoda, 2000). Two genes containing the Mei4p recognition element (see text under “Not all targets of the Mei4p transcription factor…“) show a spike in transcription at 3 h, whereas seven show a peak at 5 h (bottom left). Only two genes fit none of the regulatory paradigms defined to date (bottom middle).