In spliceosome assembly, the 5’ splice site is initially recognized by U1snRNA. U1 leaves the spliceosome during the assembly process, therefore other factors contribute to maintenance of 5’ splice site identity as it is loaded into the catalytic site. Recent structural data suggest that human tri-snRNP 27K (SNRP27) M141 and SNU66 H734 interact to stabilize the U4/U6 quasi-pseudo knot at the base of the U6 snRNA ACAGAGA box in pre-B complex. Previously, we found that mutations in C. elegans at SNRP-27 M141 promote changes in alternative 5’ss usage. We tested whether the potential interaction between SNRP-27 M141 and SNU-66 H765 (the C. elegans equivalent position to human SNU66 H734) contributes to maintaining 5’ splice site identity during spliceosome assembly. We find that SNU-66 H765 mutants promote alternative 5’ splice site usage. Many of the alternative 5’ splicing events affected by SNU-66(H765G) overlap with those affected SNRP-27(M141T). Double mutants of snrp-27(M141T) and snu-66(H765G) are homozygous lethal. We hypothesize that mutations at either SNRP-27 M141 or SNU-66 H765 allow the spliceosome to load an alternative 5’ splice sites into the active site. Tests with mutant U1 snRNA and swapped 5’ splice sites indicate that the ability of SNRP-27 M141 and SNU-66 H765 mutants to affect a particular 5’ splice alternative splicing event is dependent on both the presence of a weaker consensus 5’ss nearby and potentially nearby splicing factor binding sites. Our findings confirm a new role for the C-terminus of SNU-66 in maintenance of 5’ splice site identity during spliceosome assembly.

中文翻译：

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SNU66 在剪接体组装过程中维持 5' 剪接位点身份的作用

在剪接体组装中，5' 剪接位点最初被 U1snRNA 识别。U1 在组装过程中离开剪接体，因此当它加载到催化位点时，其他因素有助于维持 5' 剪接位点身份。最近的结构数据表明，人 tri-snRNP 27K (SNRP27) M141 和 SNU66 H734 相互作用，稳定前 B 复合物中 U6 snRNA ACAGAGA 盒底部的 U4/U6 准伪结。此前，我们发现线虫中 SNRP-27 M141 的突变促进了替代 5's 使用的变化。我们测试了 SNRP-27 M141 和 SNU-66 H765（线虫与人类 SNU66 H734 的等效位置）之间的潜在相互作用是否有助于在剪接体组装过程中维持 5' 剪接位点同一性。我们发现 SNU-66 H765 突变体促进替代 5' 剪接位点的使用。许多受 SNU-66(H765G) 影响的选择性 5' 剪接事件与受影响的 SNRP-27(M141T) 重叠。snrp-27(M141T) 和 snu-66(H765G) 的双突变体是纯合致死的。我们假设 SNRP-27 M141 或 SNU-66 H765 的突变允许剪接体将替代的 5' 剪接位点加载到活性位点中。对突变体 U1 snRNA 和交换 5' 剪接位点的测试表明，SNRP-27 M141 和 SNU-66 H765 突变体影响特定 5' 剪接选择性剪接事件的能力取决于附近是否存在较弱的共有 5'ss以及可能附近的剪接因子结合位点。我们的研究结果证实了 SNU-66 C 末端在剪接体组装过程中维持 5' 剪接位点身份的新作用。