Naguleswaran Arunasalam, Gunasekera Kapila, Schimanski Bernd, Heller Manfred, Hemphill Andrew, Ochsenreiter Torsten, Roditi Isabel
Institute of Cell Biology, University of Bern, Bern, Switzerland.
Department of Clinical Research, University of Bern, Bern, Switzerland.
mBio. 2015 Mar 17;6(2):e00114. doi: 10.1128/mBio.00114-15.
TbRRM1 of Trypanosoma brucei is a nucleoprotein that was previously identified in a search for splicing factors in T. brucei. We show that TbRRM1 associates with mRNAs and with the auxiliary splicing factor polypyrimidine tract-binding protein 2, but not with components of the core spliceosome. TbRRM1 also interacts with several retrotransposon hot spot (RHS) proteins and histones. RNA immunoprecipitation of a tagged form of TbRRM1 from procyclic (insect) form trypanosomes identified ca. 1,500 transcripts that were enriched and 3,000 transcripts that were underrepresented compared to cellular mRNA. Enriched transcripts encoded RNA-binding proteins, including TbRRM1 itself, several RHS transcripts, mRNAs with long coding regions, and a high proportion of stage-regulated mRNAs that are more highly expressed in bloodstream forms. Transcripts encoding ribosomal proteins, other factors involved in translation, and procyclic-specific transcripts were underrepresented. Knockdown of TbRRM1 by RNA interference caused widespread changes in mRNA abundance, but these changes did not correlate with the binding of the protein to transcripts, and most splice sites were unchanged, negating a general role for TbRRM1 in splice site selection. When changes in mRNA abundance were mapped across the genome, regions with many downregulated mRNAs were identified. Two regions were analyzed by chromatin immunoprecipitation, both of which exhibited increases in nucleosome occupancy upon TbRRM1 depletion. In addition, subjecting cells to heat shock resulted in translocation of TbRRM1 to the cytoplasm and compaction of chromatin, consistent with a second role for TbRRM1 in modulating chromatin structure.
Trypanosoma brucei, the parasite that causes human sleeping sickness, is transmitted by tsetse flies. The parasite progresses through different life cycle stages in its two hosts, altering its pattern of gene expression in the process. In trypanosomes, protein-coding genes are organized as polycistronic units that are processed into monocistronic mRNAs. Since genes in the same unit can be regulated independently of each other, it is believed that gene regulation is essentially posttranscriptional. In this study, we investigated the role of a nuclear RNA-binding protein, TbRRM1, in the insect stage of the parasite. We found that TbRRM1 binds nuclear mRNAs and also affects chromatin status. Reduction of nuclear TbRRM1 by RNA interference or heat shock resulted in chromatin compaction. We propose that TbRRM1 regulates RNA polymerase II-driven gene expression both cotranscriptionally, by facilitating transcription and efficient splicing, and posttranscriptionally, via its interaction with nuclear mRNAs.
布氏锥虫的TbRRM1是一种核蛋白，先前在对布氏锥虫剪接因子的搜索中被鉴定出来。我们发现TbRRM1与mRNA以及辅助剪接因子多嘧啶序列结合蛋白2相关联，但不与核心剪接体的组分相关联。TbRRM1还与几种逆转座子热点（RHS）蛋白和组蛋白相互作用。从原循环（昆虫）形式的锥虫中对带标签形式的TbRRM1进行RNA免疫沉淀，鉴定出约1500种转录本富集，与细胞mRNA相比，3000种转录本代表性不足。富集的转录本编码RNA结合蛋白，包括TbRRM1本身、几种RHS转录本、具有长编码区的mRNA以及在血流形式中表达更高的高比例阶段调节mRNA。编码核糖体蛋白、参与翻译的其他因子以及原循环特异性转录本的转录本代表性不足。通过RNA干扰敲低TbRRM1导致mRNA丰度发生广泛变化，但这些变化与该蛋白与转录本的结合无关，并且大多数剪接位点未改变，否定了TbRRM1在剪接位点选择中的一般作用。当将mRNA丰度的变化映射到整个基因组时，鉴定出许多mRNA下调的区域。通过染色质免疫沉淀分析了两个区域，在TbRRM1缺失时，这两个区域的核小体占有率均增加。此外，对细胞进行热休克导致TbRRM1易位至细胞质并使染色质压缩，这与TbRRM1在调节染色质结构中的第二个作用一致。
导致人类昏睡病的寄生虫布氏锥虫由采采蝇传播。该寄生虫在其两个宿主中经历不同的生命周期阶段，在此过程中改变其基因表达模式。在锥虫中，蛋白质编码基因被组织成多顺反子单元，这些单元被加工成单顺反子mRNA。由于同一单元中的基因可以相互独立调节，因此认为基因调节基本上是转录后调节。在本研究中，我们研究了一种核RNA结合蛋白TbRRM1在寄生虫昆虫阶段的作用。我们发现TbRRM1结合核mRNA并影响染色质状态。通过RNA干扰或热休克降低核TbRRM1会导致染色质压缩。我们提出TbRRM1通过促进转录和有效剪接在转录共调控水平上调节RNA聚合酶II驱动的基因表达，并通过其与核mRNA的相互作用在转录后水平上进行调节。
