Ustyantsev I G, Borodulina O R, Kramerov D A
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia.
Mol Biol (Mosk). 2024 May-Jun;58(3):437-447.
SINEs are mobile genetic elements of multicellular eukaryotes that arose during evolution from various tRNAs, as well as from 5S rRNA and 7SL RNA. Like the genes of these RNAs, SINEs are transcribed by RNA polymerase III. The transcripts of some mammalian SINEs have the capability of AAUAAA-dependent polyadenylation, which is unique for transcript generated by RNA polymerase III. Despite a certain similarity with canonical polyadenylation of mRNAs (transcripts of RNA polymerase II), these processes apparently differ significantly. The purpose of this work is to evaluate how important for polyadenylation of SINE transcripts are proteins of the CPSF complex formed by mPSF and mCF subcomplexes which direct mRNA polyadenylation. In HeLa cells, siRNA knockdowns of the CPSF components were carried out, after which the cells were transfected with plasmid constructs containing SINEs. A decrease in polyadenylation of the SINE transcripts as a result of the knockdown of the proteins was evaluated by Northern-hybridization. It turned out that the CPSF components, such as Wdr33 and CPSF30, contributed to the polyadenylation of SINE transcriptions, while the knockdown of CPSF100, CPSF73, and symplekin did not reduce the polyadenylation of these transcripts. Wdr33 and CPSF30, along with the CPSF160 and Fip1 previously studied, are components of the subcomplex mPSF responsible for mRNA polyadenylation. Thus, the available data suggest the importance of all mPSF proteins for polyadenylation of SINE transcripts. At the same time, CPSF100, CPSF73, and symplekin, forming the subcomplex mCF, are responsible for the cleavage of pre-mRNA; therefore, their non-participation in the polyadenylation of SINE transcriptions seems quite natural.
短散在核元件（SINEs）是多细胞真核生物中的可移动遗传元件，在进化过程中起源于各种转运RNA（tRNA），以及5S核糖体RNA（rRNA）和7SL RNA。与这些RNA的基因一样，SINEs由RNA聚合酶III转录。一些哺乳动物SINEs的转录本具有依赖于AAUAAA的多聚腺苷酸化能力，这对于RNA聚合酶III产生的转录本来说是独特的。尽管与信使核糖核酸（mRNA，RNA聚合酶II的转录本）的经典多聚腺苷酸化有一定相似性，但这些过程显然有很大差异。这项工作的目的是评估由指导mRNA多聚腺苷酸化的mPSF和mCF亚复合体形成的CPSF复合体的蛋白质对SINE转录本的多聚腺苷酸化有多重要。在HeLa细胞中，对CPSF组分进行小干扰RNA（siRNA）敲低，之后用含有SINEs的质粒构建体转染细胞。通过Northern杂交评估由于蛋白质敲低导致的SINE转录本多聚腺苷酸化的减少。结果发现，CPSF组分，如Wdr33和CPSF30，有助于SINE转录的多聚腺苷酸化，而敲低CPSF100、CPSF73和共生蛋白并没有降低这些转录本的多聚腺苷酸化。Wdr33和CPSF30，连同先前研究的CPSF160和Fip1，是负责mRNA多聚腺苷酸化的mPSF亚复合体的组分。因此，现有数据表明所有mPSF蛋白对SINE转录本的多聚腺苷酸化都很重要。同时，形成mCF亚复合体的CPSF100、CPSF73和共生蛋白负责前体mRNA的切割；因此，它们不参与SINE转录的多聚腺苷酸化似乎很自然。
