Study of the RNA splicing kinetics via in vivo 5-EU labeling.

Autor:	Bolikhova AK; Center of Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo 121205, Russia.; A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia., Buyan AI; Institute of Protein Research, Russian Academy of Sciences, Pushchino 142290, Russia.; Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia., Mariasina SS; A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia.; Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia.; Faculty of Fundamental Medicine, Lomonosov Moscow State University, Moscow 119991, Russia., Rudenko AY; A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia., Chekh DS; Faculty of Biology, Lomonosov Moscow State University, Moscow 119991, Russia., Mazur AM; Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia., Prokhortchouk EB; Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia., Dontsova OA; Center of Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo 121205, Russia.; A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia.; Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia.; Department of Functioning of Living Systems, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia., Sergiev PV; Center of Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo 121205, Russia petya@genebee.msu.ru.; A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia.; Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia.; Institute of Functional Genomics, Lomonosov Moscow State University, Moscow 119991, Russia.
Jazyk:	angličtina
Zdroj:	RNA (New York, N.Y.) [RNA] 2024 Sep 16; Vol. 30 (10), pp. 1356-1373. Date of Electronic Publication: 2024 Sep 16.
DOI:	10.1261/rna.079937.123
Abstrakt:	Splicing is an important step of gene expression in all eukaryotes. Splice sites might be used with different efficiency, giving rise to alternative splicing products. At the same time, splice sites might be used at a variable rate. We used 5-ethynyl uridine labeling to sequence a nascent transcriptome of HeLa cells and deduced the rate of splicing for each donor and acceptor splice site. The following correlation analysis showed a correspondence of primary transcript features with the rate of splicing. Some dependencies we revealed were anticipated, such as a splicing rate decrease with a decreased complementarity of the donor splice site to U1 and acceptor sites to U2 snRNAs. Other dependencies were more surprising, like a negative influence of a distance to the 5' end on the rate of the acceptor splicing site utilization, or the differences in splicing rate between long, short, and RBM17-dependent introns. We also observed a deceleration of last intron splicing with an increase of the distance to the poly(A) site, which might be explained by the cooperativity of the splicing and polyadenylation. Additional analysis of splicing kinetics of SF3B4 knockdown cells suggested the impairment of a U2 snRNA recognition step. As a result, we deconvoluted the effects of several examined features on the splicing rate into a single regression model. The data obtained here are useful for further studies in the field, as they provide general splicing rate dependencies as well as help to justify the existence of slowly removed splice sites. (© 2024 Bolikhova et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
Databáze:	MEDLINE
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