Activity of METTL4 Methyltransferase Is Crucial for Maintaining Optimal Splicing Efficiency in HeLa S3 Cells

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Anastasiia K. Bolikhova^1,2,3,a*#, Andrey I. Buyan^3,4#, Maria A. Khokhlova^1,3, Sofia S. Mariasina^2,3,5, Anton R. Izzi^1,6, Alexander Y. Rudenko², Marina V. Serebryakova², Alexander M. Mazur⁷, Olga A. Dontsova^1,2,3,8, and Petr V. Sergiev^1,2,3

¹Skolkovo Institute of Science and Technology, Center for Life Sciences, 121205 Skolkovo, Russia

²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

³Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia

⁴Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia

⁵Research and Educational Resource Center “Pharmacy”, RUDN University, 117198 Moscow, Russia

⁶Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119991 Moscow, Russia

⁷Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia

⁸Department of Functioning of Living Systems, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

^* To whom correspondence should be addressed.

^# These authors contributed equally to the work.

Received: August 8, 2025; Revised: November 11, 2025; Accepted: November 13, 2025

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Methyltransferases that modify spliceosomal small nuclear RNAs (snRNAs) play a crucial role in the cell by ensuring proper maturation of snRNAs, which is essential for optimal function of spliceosome. In this study, we investigated the enzyme METTL4, which catalyzes N6-methylation of 2′-O-methyladenosine at position 30 of U2 snRNA. Function of both the protein and the modification in splicing remains unclear. We demonstrated that inactivation of the METTL4 gene in HeLa S3 cells leads to significant changes in alternative splicing, general slowdown in spliceosome activity, and intron accumulation. In the cells lacking METTL4, expression of the set of genes associated with ribosomal RNA maturation is reduced, and the number of coilin-positive structures, most likely Cajal bodies, is decreased in the nuclei of these cells.

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KEY WORDS: splicing, small nuclear RNAs (snRNAs), METTL4, U2 snRNA, methylation

DOI: 10.1134/S0006297925602382

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