Transcription-independent functions of an RNA polymerase II subunit, Rpb2, during genome rearrangement in the ciliate, Oxytricha trifallax

Abstract

The RNA polymerase II (Pol-II) holoenzyme, responsible for messenger RNA production, typically consists of 10-12 subunits. Our laboratory previously demonstrated that maternally deposited, long, noncoding, template RNAs are essential for programmed genome rearrangements in the ciliate Oxytricha trifallax. Here we show that such RNAs are bidirectionally transcribed and transported to the zygotic nucleus. The gene encoding the second-largest subunit of Pol-II, Rpb2, has undergone gene duplication, and the two paralogs, Rpb2-a and -b, display different expression patterns. Immunoprecipitation of double-stranded RNAs identified an association with Rpb2-a. Through immunoprecipitation and mass spectrometry, we show that Rpb2-a in early zygotes appears surprisingly unassociated with other Pol II subunits. A partial loss of function of Rpb2-a leads to an increase in expression of transposons and other germline-limited satellite repeats. We propose that evolutionary divergence of the Rpb2 paralogs has led to acquisition of transcription-independent functions during sexual reproduction that may contribute to the negative regulation of germline gene expression.

Keywords: RNA polymerase; genome integrity; noncoding RNAs; transcription.

Figure 1

Bidirectional transcription at 12 hr postmating. (A) Chromatin immunoprecipitation (IP) with IgG (negative control) or Rpb1 antibody at 12 and 24 hr postmixing, followed by quantitative PCR (qPCR) for the indicated contigs or gene loci. The graphs are represented as percentage of IP relative to the input. (B) RNA-IP using anti-Rpb1 antibody, followed by RT-qPCR for indicated transcripts. (C and D) Mapping of Rpb1 ChIP-seq reads for (C) Contig 22209.0 (TEBP-β) and (D) Contig 451.1 (ribosomal RNA) at 12 hr. The intensity of the signal (green) represents the number of ChIP-seq reads mapping to that region; I = intron in panel (C).

Figure 2

Localization and evolutionary duplication of Rpb2. (A) Cells fixed at indicated times were stained using the Rpb2 antibody (green) and DAPI (blue). Rpb2 localizes to micronuclei (I) at all stages during conjugation and to the zygotic macronucleus (Z) most strikingly at 18 hr. No visible signal is seen in the parental macronuclei (P) or at 0 hr. (B) RT-PCR analysis of Rpb2-a and Rpb2-b during development. (C) Pfam protein domain analysis of human Rpb2 (H.sap) and Oxytricha Rpb2-a and Rpb2-b. The domain annotation is RNA_Pol_Rpb- followed by unique identifiers shown. Protein domains are drawn to scale. (D) Maximum-likelihood phylogenetic analysis of Rpb2 from O. trifallax (O.tri Rpb2-a and Rpb2-b), T. thermophila (T.the), P. tetraurelia (P.tet), Plasmodium falciparum (P.fal), E. crassus (E.cra), D. melanogaster (D.mel), M. musculus (M.mus), H. sapiens (H.sap), S. cerevisiae (S.cer), S. pombe (S.pom), D. rerio (D.rer). Urostyla sp. (Uro.sp_Rpb2a and Rpb2b), O. nova (O.nov_Rpb2-a and -b), S. lemnae (S.lem_Rpb2-a and -b), S. histriomuscorum (S.his_Rpb2-a and -b), Paraurostyla sp. (Par.sp_Rpb2-a and -b), and Laurentialla sp. (Lau.sp_Rpb2-a and -b).

Figure 3

Rpb2-a is not associated with the Rpb1 complex at 18 hr postmixing. (A) Peptide spectrum match (PSM) scoring function for IgG compared to two replicates of Rpb2-IP at 18 hr. (B) Rpb1-IP at 12 and 18 hr postmixing, followed by Western blot analysis of Rpb2 and Rpb1, indicating the abundant presence of Rpb2 in the Pol-II complex at 12 and 18 hr. (C) Western hybridization of cell extracts from 12 hr postmixing, probed with Rpb1 or Rpb2 antibodies and exposed overnight to demonstrate the specificity of the antibody.

Figure 4

Rpb2-a or Otiwi-1 knockdown affects germline gene expression. (A and B) Rpb2-a (A) and Otiwi-1 (B) knockdown using phosphorothioate-modified antisense oligonucleotides, followed by mRNA expression analysis via RT-qPCR for the indicated genes at 24 hr postmixing (see text for gene names), relative to mitochondrial rRNA (mito-rRNA) as control uninjected cells. * denotes significant changes relative to control; P-values are noted in the text. Labels 380bp and 170bp refer to MIC-limited satellite repeats (Bracht et al. 2012). The error bars represent standard error of the mean (SEM). (C) mRNA half-life (in hours) for Dcl-1, Otiwi-1, and Spt5 calculated using an Actinomycin D treatment pulse/chase experiment.