A meiosis-specific Spt5 homolog involved in non-coding transcription

Abstract

Spt5 is a conserved and essential transcriptional regulator that binds directly to RNA polymerase and is involved in transcription elongation, polymerase pausing and various co-transcriptional processes. To investigate the role of Spt5 in non-coding transcription, we used the unicellular model Paramecium tetraurelia. In this ciliate, development is controlled by epigenetic mechanisms that use different classes of non-coding RNAs to target DNA elimination. We identified two SPT5 genes. One (STP5v) is involved in vegetative growth, while the other (SPT5m) is essential for sexual reproduction. We focused our study on SPT5m, expressed at meiosis and associated with germline nuclei during sexual processes. Upon Spt5m depletion, we observed absence of scnRNAs, piRNA-like 25 nt small RNAs produced at meiosis. The scnRNAs are a temporal copy of the germline genome and play a key role in programming DNA elimination. Moreover, Spt5m depletion abolishes elimination of all germline-limited sequences, including sequences whose excision was previously shown to be scnRNA-independent. This suggests that in addition to scnRNA production, Spt5 is involved in setting some as yet uncharacterized epigenetic information at meiosis. Our study establishes that Spt5m is crucial for developmental genome rearrangements and necessary for scnRNA production.

Figure 1.

Spt5 proteins in Paramecium. (A) Predicted protein domains (colored boxes) for Paramecium tetraurelia, Tetrahymena thermophila, Homo sapiens, Saccharomyces cerevisiae and Arabidopsis thaliana Spt5 proteins. (B) Neighbor-joining tree of Spt5 proteins from ciliates based on the alignment of entire protein sequences (461 positions in the final dataset) and the following parameters: bootstrap 1000 (bootstrap values displayed next to the branches) and Poisson correction. Ptet, P. tetraurelia; Pbi, Paramecium biaurelia; Psex, Paramecium sexaurelia; Pcaud, Paramecium caudatum; Ot, Oxytricha trifallax; Tt, T. thermophila, human (Hs) and arabidopsis (At). (C) Autogamy time-course of gene expression for P. tetraurelia SPT5 (GSPATG00013468001, GSPATG00023145001), DCL2 (GSPATG00008494001), DCL3 (GSPATG00027456001) and SPO11 (GSPATG00009108001). The Y-axis shows the log2 signals from custom microarrays (55,59).

Figure 2.

Localization of Spt5-GFP fusion proteins. For both SPT5v-GFP and SPT5m-GFP transgenes, representative images illustrate different developmental stages. Panels a and i show vegetative cells. Successive stages of autogamy are shown in the following panels: panels b and j—meiotic crescent stage; panels c and k—cells with eight haploid nuclei resulting from meiosis II (only six nuclei are visible in panel C); panels d, e, l and m—fragmentation of old MAC; panel L—first division of the zygotic nucleus; panel M—second division of the zygotic nucleus; panels f and n—early MAC development; panels g, h, o and p—late MAC development. In all panels, white arrows point at MICs (some were omitted when MICs were not clearly distinguishable by DAPI staining), white arrowheads indicate new MACs. Yellow arrowheads in panels L and M point to division products of the zygotic nucleus. The Spt5v-GFP fusion (left side) localizes to old, then new MACs. The Spt5m-GFP (right side) localizes to meiotic MICs, the zygotic nucleus as well as products of division of the zygotic nucleus.

Figure 3.

Analysis of sRNA populations in SPT5m-RNAi cells. (A) Gel electrophoresis of sRNA from SPT5m-silenced cells and control silencing. Total RNA samples corresponding to the T0, T5, T10 and T15 time-points were run on a denaturing 15% polyacrylamide-urea gel. After electrophoresis the gel was stained with SYBR Gold (Invitrogen). M: DNA Low Molecular Weight Marker (USB). The ∼25 nt signal that corresponds to the fraction of scnRNAs (29) is labeled. In the control, at the T15 time-point, additional bands corresponding to 26–30 nt iesRNAs are present (indicated by a bracket). In SPT5m-silenced samples neither scnRNAs nor iesRNAs can be seen. (B) Small RNA libraries corresponding to early and late development time-points were sequenced and mapped to the reference genomes (Paramecium tetraurelia MAC reference genome and MAC+IES reference genome). Results obtained for a control culture (cells silenced for ND7 gene expression) are shown in the left hand panel, SPT5m-silencing in the middle panel and DCL2/3-silencing (from (30)) in the right hand panel. Stacked bar plots show the normalized number of sRNA reads that match the MAC genome (blue), annotated IESs (yellow) or were not mapped (gray). (C) Read counts obtained during sequencing of small RNA libraries corresponding to early and late development time-points were normalized. Bar plots show the relative amounts of sRNA reads for control (white), SPT5-RNAi (gray) and DCL2/3-RNAi (black, for details of see (30)).The left hand panel corresponds to scnRNA and shows the number of 25 nt reads divided by the number of 23 nt siRNA reads for each sample. Right hand panel shows iesRNA reads (26–29 nt) in relation to 23 nt reads. Since iesRNAs are supposed to be produced by Dcl5 from excised IESs (30), their appearance in the late sample could be reduced owing to reduced IES excision in SPT5 and DCL2/3-RNAi.

Figure 4.

Analysis of IES excision in SPT5m-silenced cells. (A) Retention score distribution determined by re-sequencing DNA extracted from a cell fraction enriched in new MACs, after SPT5m silencing. IESs with a given SPT5m retention score that are sensitive to DCL2/3, TFIIS4 or EZL1 as determined using published data (34,37,38) are indicated. Bar coloring: purple, DCL2/3 + TFIIS + EZL1; yellow, TFIIS4 + EZL1; red, EZL1. (B) Venn diagram showing significantly retained IESs after SPT5m, EZL1, TFIIS4, DCL2/3 or DCL5 silencing. Details can be found in Supplementary Table S4.