In higher eukaryotes, most genes contain introns. After transcription is complete, the removal of introns from mRNA should be done by splicing to generate mature and translationally active mRNA. This process is carried out and stimulated by the spliceosome. The main spliceosome contains five types of snRNA (small nuclear RNA), i.e., U1, U2, U4, U5, and U6, as well as the protein factors that interact with them [1]. Among them, U6 snRNA is at the core and most conservative position. It is located at the catalytic center of the spliceosome, which is essential for the catalytic activity of the spliceosome [2]. At the same time, it was also found that U6 is highly modified in metamorphosis, including the modifications taking place in the 5’ terminal γ-monomethylation capping and 3-oligomeric uridine, as well as the intermediate 2-O-methylated, pseudouridine, m2G and m6A, etc. [3,4]. After nearly 40 years of research, researchers have revealed the biological production process of U6 and its functional mechanism in splicing, but the regulatory mechanism of U6 modification and its function in mRNA splicing is still not well understood.

 

Previous studies have found that in adult mammals, the testis has the tissue with the highest transcriptional activity and the most abundant alternative splicing [5], which implies that the RNA splicing machinery of the testes may be highly active. Consistent with this, spermatogenesis is regulated by specific spatiotemporal expression of genes - each stage of spermatogenic cell development has a specific gene regulation and alternative splicing of genes related to signal transduction. Therefore, the testicular tissues should be an ideal system for studying mammalian U6 modification and its effect on mRNA splicing.

 

On February 3rd, 2020, Mo-Fang Liu’s research team, affiliated with the Center for Excellence in Molecular Cell Science, CAS, published an article titled “LARP7-Mediated U6 snRNA Modification Ensures Splicing Fidelity and Spermatogenesis in Mice” in Molecular Cell. The publication reports that RNA-binding protein LARP7 mediates the 2-O-methylation modification of U6 by promoting the interaction between U6 snRNA and the box C/D snoRNP with catalytic activity for RNA methylation - further proving that this process is essential for mRNA splicing fidelity and spermatogenesis in the spermatogenic cells of mice.

 

 

 

In this study, the members of Mo-Fang Liu’s research team used mouse testes as a model system to explore the regulatory mechanism of U6 snRNA modification and its function in mRNA splicing. They found that the LARP7 protein, which is highly expressed in the testes, is essential for the 2-O-methylation modification of U6 in spermatogenic cells. It has been found that RNA-binding protein LARP7 inhibits the elongation of RNA polymerase II transcription by binding to 7SK RNA [6,7]. Some studies show that Larp7 gene mutation is associated with human Alazami syndrome [8]. Further mechanism research reveals that LARP7 binds to U6 and snoRNA simultaneously to assist U6 loading onto box C/D snoRNP, thus promoting the methyltransferase FBL in box C/D snoRNP to achieve 2-O-methylation modification of it. More importantly, they found that LARP7-mediated U6 2-O-methylation modification is vital for precise mRNA splicing and spermatogenesis in mouse spermatogenic cells.

 

The animal experiments indicate that specifically knocking out the Larp7 gene in the germ cells causes infertility in male mice (the Larp7 gene conditional knockout mice came from Cyagen Biosciences).

 

This study revealed the regulatory mechanism of U6 snRNA modification and proved for the first time that U6 2-O-methylation modification is essential for precise splicing of mammalian mRNA and spermatogenesis in mouse spermatogenic cells.

 

In addition, both Gunter Meister’s research team, from the University of Regensburg, Germany, and Utz Fischer’s research team, from the University of Würzburg, also partnered with Mo-Fang Liu’s research team to publish an article titled “The Alazami Syndrome-associated Protein LARP7 Guides U6 small nuclear RNA Modification and Contributes to Splicing Robustness”. This article reports that the LARP7 in human cells can also mediate the 2-O-methylation modification of U6 by binding U6 and snoRNA. More importantly, in a patient with Alazami syndrome carrying a Larp7 mutation, they detected reduced 2-O-methylation modification of U6 and abnormal mRNA splicing.

 

Together, these research works prove that LARP7 mediates U6 modification and affects precise mRNA splicing in mammals. Their findings not only reveal the regulatory mechanism of U6 modification and its effect on mRNA splicing, but also help to analyze the pathogenesis of male infertility, Alazami syndrome, etc., and provide a theoretical basis and method for the diagnosis and treatment of related diseases.

 

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Original link:

https://doi.org/10.1016/j.molcell.2020.01.002

https://doi.org/10.1016/j.molcel.2020.01.001

 

References：

1. Patel, A.A., and Steitz, J.A. (2003). Splicing double: insights from the second spliceosome. Nat Rev Mol Cell Bio 4, 960-970.

2. Didychuk, A.L., Butcher, S.E., and Brow, D.A. (2018). The life of U6 small nuclear RNA, from cradle to grave. RNA 24, 437-460.

3. Epstein, P., Reddy, R., Henning, D., and Busch, H. (1980). The nucleotide sequence of nuclear U6 (4.7 S) RNA. J Biol Chem 255, 8901-8906.

4. Harada, F., Kato, N., and Nishimura, S. (1980). The nucleotide sequence of nuclear 4.8S RNA of mouse cells. Biochem Biophys Res Commun 95, 1332-1340.

5. White-Cooper, H., and Davidson, I. (2011). Unique aspects of transcription regulation in male germ cells. Cold Spring Harb Perspect Biol 3 a002626.

6. Krueger, B.J., Jeronimo, C., Roy, B.B., Bouchard, A., Barrandon, C., Byers, S.A., Searcey, C.E., Cooper, J.J., Bensaude, O., and Cohen, E.A., et al. (2008). LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated. Nucleic Acids Res 36, 2219-2229.

7. Markert, A., Grimm, M., Martinez, J., Wiesner, J., Meyerhans, A., Meyuhas, O., Sickmann, A., and Fischer, U. (2008). The La-related protein LARP7 is a component of the 7SK ribonucleoprotein and affects transcription of cellular and viral polymerase II genes. EMBO Rep 9, 569-575.

8. Alazami, A.M., Al-Owain, M., Alzahrani, F., Shuaib, T., Al-Shamrani, H., Al-Falki, Y.H., Al-Qahtani, S.M., Alsheddi, T., Colak, D., and Alkuraya, F.S. (2012). Loss of function mutation in LARP7, chaperone of 7SK ncRNA, causes a syndrome of facial dysmorphism, intellectual disability, and primordial dwarfism. Hum Mutat 33, 1429-1434.

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