Gopanenko Alexander V, Malygin Alexey A, Tupikin Alexey E, Laktionov Pavel P, Kabilov Marsel R, Karpova Galina G
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Department of Molecular Biology, Novosibirsk State University, Novosibirsk 630090, Russia.
Nucleic Acids Res. 2017 Sep 6;45(15):9121-9137. doi: 10.1093/nar/gkx559.
Ribosomal proteins are involved in many cellular processes through interactions with various RNAs. Here, applying the photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation approach to HEK293 cells overproducing ribosomal protein (rp) eS1, we determined the products of RNU5A-1 and RNU11 genes encoding U5 and U11 snRNAs as the RNA partners of ribosome-unbound rp eS1. U11 pre-snRNA-associated rp eS1 was revealed in the cytoplasm and nucleus where rp eS1-bound U11/U12 di-snRNP was also found. Utilizing recombinant rp eS1 and 4-thiouridine-containing U11 snRNA transcript, we identified an N-terminal peptide contacting the U-rich sequence in the Sm site-containing RNA region. We also showed that the rp eS1 binding site on U11 snRNA is located in the cleft between stem-loops I and III and that its structure mimics the respective site on the 18S rRNA. It was found that cell depletion of rp eS1 leads to a decrease in the splicing efficiency of minor introns and to an increase in the level of U11 pre-snRNA with the unprocessed 3' terminus. Our findings demonstrate the engagement of human rp eS1 in events related to the U11 snRNA processing and to minor-class splicing. Contacts of rp eS1 with U5 snRNA in the minor pre-catalytic spliceosome are discussed.
核糖体蛋白通过与各种RNA相互作用参与许多细胞过程。在此，我们将光活化核糖核苷增强的交联和免疫沉淀方法应用于过量表达核糖体蛋白（rp）eS1的HEK293细胞，确定了编码U5和U11小核仁RNA（snRNA）的RNU5A-1和RNU11基因的产物为核糖体未结合的rp eS1的RNA伴侣。在细胞质和细胞核中发现了与U11前体snRNA相关的rp eS1，在那里也发现了与rp eS1结合的U11/U12双小核核糖核蛋白颗粒（di-snRNP）。利用重组rp eS1和含4-硫尿苷的U11 snRNA转录本，我们鉴定出一个与含Sm位点的RNA区域中富含U的序列接触的N端肽段。我们还表明，U11 snRNA上的rp eS1结合位点位于茎环I和III之间的裂隙中，其结构模仿18S核糖体RNA（rRNA）上的相应位点。研究发现，细胞中rp eS1的缺失导致小内含子剪接效率降低，以及具有未加工3'末端的U11前体snRNA水平升高。我们的研究结果证明了人类rp eS1参与了与U11 snRNA加工和小类剪接相关的事件。本文还讨论了rp eS1在小前催化剪接体中与U5 snRNA的相互作用。
