The testis-specific double bromodomain-containing protein BRDT forms a complex with multiple spliceosome components and is required for mRNA splicing and 3'-UTR truncation in round spermatids

Abstract

Members of the BET (bromodomain and extra terminal motif) family of proteins have been shown to be chromatin-interacting regulators of transcription. We previously generated a mutation in the testis-specific mammalian BET gene Brdt (bromodomain, testis-specific) that yields protein lacking the first bromodomain (BRDT(ΔBD1)) and observed disrupted spermiogenesis and male sterility. To determine whether BRDT(ΔBD1) protein results in altered transcription, we analyzed the transcriptomes of control versus Brdt(ΔBD1/ΔBD1) round spermatids. Over 400 genes showed statistically significant differential expression, and among the up-regulated genes, there was an enrichment of RNA splicing genes. Over 60% of these splicing genes had transcripts that lacked truncation of their 3'-untranslated region (UTR) typical of round spermatids. We selected four of these genes to characterize: Srsf2, Ddx5, Hnrnpk and Tardbp. The 3'-UTRs of Srsf2, Ddx5 and Hnrnpk mRNAs were longer in mutant round spermatids and resulted in reduced protein levels. Tardbp was transcriptionally up-regulated and a splicing shift toward the longer variant was observed. All four splicing proteins were found to complex with BRDT in control and mutant testes. We thus suggest that, along with modulating transcription, BRDT modulates gene expression as part of the splicing machinery. These modulations alter 3'-UTR processing in round spermatids; importantly, the BD1 is essential for these functions.

Figure 1.

Microarray analysis of RNA from purified control and Brdt^ΔBD1/ΔBD1 mutant round spermatids reveals genes that are both up-regulated and down-regulated in the mutant cells. (A) Heat map of the eight microarrays showing two groups of genes, one up-regulated and one down-regulated in mutant round spermatids. (B) A total of 415 genes exhibited statistically significant (adjPval ≤0.05) altered expression (logFC ≥1 or ≤−1) in mutant round spermatids. When the criteria were relaxed to include all differential expression, 1478 genes with altered expression were observed.

Figure 2.

Detailed examination of Affymetrix probe sets yields unexpected information about mRNA processing as well as expected changes in total transcription. (A and B) Cartoon schematics of two genes, Srsf2 and Tardbp, showing the location of the Affymetrix probes, designed qPCR primers and northern blot probes. The qPCR primer location mirrors the positions of the Affymetrix probes, and northern blot probes are chosen to hybridize to all transcripts. (A) Three Affymetrix probes to the Srsf2 gene are depicted: one in the coding region, one in the proximal 3′-UTR and one in the distal 3′-UTR. The probe in the distal 3′-UTR would uniquely hybridize to the long transcript of Srsf2. In control spermatids, hybridization of the Srsf2 distal UTR probe is low compared to the coding and proximal UTR probes demonstrating lack of the Srsf2 long transcript. The long transcript is present in mutant spermatids. (B) Three Affymetrix probes to the Tardbp gene are depicted: one in the coding region, one unique to transcript variant 1 and one in the distal 3′-UTR. Tardbp transcription is up-regulated in mutant spermatids, but the possibility exists that there may be a specific up-regulation of transcript variant 1. (C) Q-PCR of four selected genes. Overall levels of transcription of Srsf2, Ddx5 and Hnrnpk are not altered in mutant round spermatids. However, the presence of normally truncated 3′-UTR sequence is increased. In contrast, overall levels of Tardbp transcription are increased in mutant round spermatids, in particular, for transcript variant 1.

Figure 3.

Loss of truncation of the 3′-UTR in mRNAs from Brdt^ΔBD1/ΔBD1 round spermatids. (A–D) Northern blot hybridization analysis of total RNA isolated from control and mutant pachytene spermatocytes and round spermatids. (A) Srsf2 long and short mRNA transcripts are present in control and mutant pachytene spermatocytes. mRNA expression is comparable in these two populations. Only short transcripts are present in control round spermatids, indicating that all Srsf2 transcripts have a truncated 3′-UTR in this cell type. Mutant spermatids express long and short Srsf2 transcripts and the total mRNA expression is comparable to that of the spermatocyte populations, indicating that there is a loss of truncation of some mRNAs. (B) Ddx5 long and short mRNA transcripts are present in control and mutant pachytene spermatocytes and control round spermatids. mRNA expression is similar in these three populations, but short transcripts make up a greater percentage of all Ddx5 transcripts in control spermatids. Mutant spermatids also express long and short transcripts, but the long transcript is much more abundant relative to the short as compared to control spermatids. (C) Hnrnpk is mostly expressed as short transcripts in control and mutant pachytene spermatocytes although there is some expression of long transcripts. There is a slight reduction in short transcripts in mutant spermatocytes. Control round spermatids only express short Hnrnpk transcripts, but mutant spermatids still express the long Hnrnpk transcripts. (D) The long transcript variant 1 of Tardbp is highly expressed in control pachytene spermatocytes along with lower expression of the shorter variants. In mutant pachytene spermatocytes, there is a general increase in expression of Tardbp mRNA, in particular, for the long variant 1 transcript. The same patterns of Tardbp expression are seen in control and mutant round spermatids. (E and F) In situ hybridization of adult control and mutant testicular sections with Srsf2 probes. (E) A probe to the coding region of Srsf2 hybridized in control and mutant pachytene spermatocytes (P) and control and mutant round spermatids (RSs). (F) A probe to the distal end of the Srsf2 3′-UTR hybridized in control and mutant pachytene spermatocytes, but only in mutant round spermatids. Control round spermatids do not expressed Srsf2 mRNA with a long 3′-UTR.

Figure 4.

Changes in protein expression correlate with alterations in 3′-UTR processing and alternative splicing. (A) Immunoblot analysis of protein extracted from whole control and Brdt^ΔBD1/ΔBD1 mutant testes of mice at post natal age 17, 22 and 24 days. At Day 17, no round spermatids are present in the testis. At Day 22 and even more so at Day 24, round spermatids compose a large proportion of the cells of the testis. SRSF2, DDX5 and HNRNPK protein expression is unchanged in Day 17 mutants. In Day 22 mutants, there is a slightly lower expression of these proteins, and this reduction in expression is even more pronounced at Day 24. The higher molecular weight band above HNRNPK is mouse IgG. Total TARDPB expression is increased relative to control expression at all time points. The ratio of longest isoform to the shortest isoforms is also increased in all mutant samples. An unknown ∼40 kDa isoform of TARDBP is weakly expressed in Day 22 control testis. Expression of this isoform is increased in the Day 22 mutant testis and its expression newly appears in the Day 17 mutant testis. (B) Immunostaining of SRSF2 in adult control and mutant testicular sections. SRSF2 protein is highly expressed in control pachytene spermatocytes and round spermatids and at a comparably high level in mutant spermatocytes. SRSF2 expression in mutant spermatids is noticeably reduced.

Figure 5.

Full-length and truncated BRDT protein can interact with SRSF2, DDX5, HNRNPK and TARDBP. (A) BRDT-containing complexes were precipitated from control and Brdt^ΔBD1/ΔBD1 mutant whole testis lysates using an anti-BRDT antibody or control rabbit IgG. Immunoblotting of the co-precipitated proteins was carried out with SRSF2, DDX5, HNRNPK and TARDBP antibodies. These four proteins all co-precipitate with both full-length and truncated BRDT protein, but not with IgG. IgG can be seen in all lanes at 55 kDa. (B) Co-immunoprecipitation using anti-SRSF2, anti-DDX5, anti-HNRNPK and anti-TARDBP antibodies and control anti-BRDT antibody and IgG with whole heterozygous Brdt^ΔBD1/+ testis lysates. Both full-length and truncated BRDT protein co-precipitates with all four proteins, but not IgG.