Norppa Antto J, Chowdhury Iftekhar, van Rooijen Laura E, Ravantti Janne J, Snel Berend, Varjosalo Markku, Frilander Mikko J
Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Molecular Systems Biology Research Group and Proteomics Unit, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Nucleic Acids Res. 2024 Apr 24;52(7):4037-4052. doi: 10.1093/nar/gkae070.
Here, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of U11/U12-65K, a known unique component of the U11/U12 di-snRNP. Both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with U11/U12-65K, which uses its N-terminal region to interact with U11 snRNP during intron recognition. Finally, while RBM41 knockout cells are viable, they show alterations in U12-type 3' splice site usage. Together, our results highlight the role of the 3'-terminal stem-loop of U12 snRNA as a dynamic binding platform for the U11/U12-65K and RBM41 proteins, which function at distinct stages of the assembly/disassembly cycle.
在此，我们鉴定出RBM41是次要剪接体的一种新型独特蛋白质成分。RBM41此前未被识别出具有细胞功能，但已被鉴定为U11/U12 - 65K的旁系同源物，U11/U12 - 65K是U11/U12双小核核糖核蛋白（di - snRNP）的一种已知独特成分。这两种蛋白质都利用其高度相似的C末端RNA识别基序（RRMs）以相当的亲和力结合到U12和U6atac小核RNA（snRNAs）的3'末端茎环结构上。我们的生物ID数据表明，RBM41独特的N末端结构域对于其与包含RNA解旋酶DHX8的复合物的结合是必需的，在主要剪接体中，DHX8驱动成熟mRNA从剪接体中释放。一致地，我们表明RBM41与切除的U12型内含子套索结构相关联，存在于U12单小核核糖核蛋白中，并在 Cajal体中富集，这共同表明RBM41在次要剪接体组装/拆卸循环的剪接后步骤中发挥作用。这与U11/U12 - 65K形成对比，U11/U12 - 65K在内含子识别过程中利用其N末端区域与U11小核核糖核蛋白相互作用。最后，虽然RBM41基因敲除细胞是有活力的，但它们在U12型3'剪接位点的使用上表现出改变。总之，我们的结果突出了U12 snRNA的3'末端茎环结构作为U11/U12 - 65K和RBM41蛋白质的动态结合平台的作用，这两种蛋白质在组装/拆卸循环的不同阶段发挥作用。
