Spliced leader trans-splicing is essential for gene expression in many eukaryotes. To elucidate the molecular mechanism of this process, we characterise the molecules associated with the Caenorhabditis elegans major spliced leader snRNP (SL1 snRNP), which donates the spliced leader that replaces the 5′ untranslated region of most pre-mRNAs. Using a GFP-tagged version of the SL1 snRNP protein SNA-1 created by CRISPR-mediated genome engineering, we immunoprecipitate and identify RNAs and protein components by RIP-Seq and mass spectrometry. This reveals the composition of the SL1 snRNP and identifies associations with spliceosome components PRP-8 and PRP-19. Significantly, we identify a novel, nematode-specific protein required for SL1 trans-splicing, which we designate SNA-3. SNA-3 is an essential, nuclear protein with three NADAR domains whose function is unknown. Mutation of key residues in NADAR domains inactivates the protein, indicating that domain function is required for activity. SNA-3 interacts with the CBC-ARS2 complex and other factors involved in RNA metabolism, including SUT-1 protein, through RNA or protein-mediated contacts revealed by yeast two-hybrid assays, localisation studies and immunoprecipitations. Our data are compatible with a role for SNA-3 in coordinating trans-splicing with target pre-mRNA transcription or in the processing of the Y-branch product of the trans-splicing reaction.

中文翻译：

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一种新型的必需转拼蛋白将线虫 SL1 snRNP 连接到 CBC-ARS2 复合物

剪接前导序列反式剪接对于许多真核生物中的基因表达至关重要。为了阐明这一过程的分子机制，我们对与秀丽隐杆线虫主要剪接前导序列 snRNP (SL1 snRNP) 相关的分子进行了表征，该分子提供了取代大多数前 mRNA 的 5' 非翻译区的剪接前导序列。使用通过 CRISPR 介导的基因组工程创建的 SL1 snRNP 蛋白 SNA-1 的 GFP 标记版本，我们通过 RIP-Seq 和质谱法免疫沉淀并鉴定 RNA 和蛋白质成分。这揭示了 SL1 snRNP 的组成，并确定了与剪接体成分 PRP-8 和 PRP-19 的关联。值得注意的是，我们鉴定了 SL1 反式剪接所需的一种新型线虫特异性蛋白，我们将其命名为 SNA-3。SNA-3 是一种必需的核蛋白，具有三个 NADAR 结构域，其功能尚不清楚。NADAR 结构域中关键残基的突变使蛋白质失活，表明结构域功能是活性所必需的。SNA-3 通过酵母双杂交测定、定位研究和免疫沉淀揭示的 RNA 或蛋白质介导的接触，与 CBC-ARS2 复合物和参与 RNA 代谢的其他因子（包括 SUT-1 蛋白）相互作用。我们的数据与 SNA-3 在协调反式剪接与靶前 mRNA 转录或处理反式剪接反应的 Y 分支产物中的作用相一致。