Gardiner Timothy J, Christov Christo P, Langley Alexander R, Krude Torsten
Department of Zoology, University of Cambridge, Cambridge CB23EJ, United Kingdom.
RNA. 2009 Jul;15(7):1375-85. doi: 10.1261/rna.1472009. Epub 2009 May 27.
Noncoding Y RNAs are required for the reconstitution of chromosomal DNA replication in late G1 phase template nuclei in a human cell-free system. Y RNA genes are present in all vertebrates and in some isolated nonvertebrates, but the conservation of Y RNA function and key determinants for its function are unknown. Here, we identify a determinant of Y RNA function in DNA replication, which is conserved throughout vertebrate evolution. Vertebrate Y RNAs are able to reconstitute chromosomal DNA replication in the human cell-free DNA replication system, but nonvertebrate Y RNAs are not. A conserved nucleotide sequence motif in the double-stranded stem of vertebrate Y RNAs correlates with Y RNA function. A functional screen of human Y1 RNA mutants identified this conserved motif as an essential determinant for reconstituting DNA replication in vitro. Double-stranded RNA oligonucleotides comprising this RNA motif are sufficient to reconstitute DNA replication, but corresponding DNA or random sequence RNA oligonucleotides are not. In intact cells, wild-type hY1 or the conserved RNA duplex can rescue an inhibition of DNA replication after RNA interference against hY3 RNA. Therefore, we have identified a new RNA motif that is conserved in vertebrate Y RNA evolution, and essential and sufficient for Y RNA function in human chromosomal DNA replication.
在一个无细胞的人类系统中，G1期晚期模板细胞核内染色体DNA复制的重建需要非编码Y RNA。Y RNA基因存在于所有脊椎动物以及一些特定的非脊椎动物中，但其功能的保守性以及功能的关键决定因素尚不清楚。在此，我们鉴定出了Y RNA在DNA复制中功能的一个决定因素，该因素在整个脊椎动物进化过程中都保持保守。脊椎动物的Y RNA能够在无细胞的人类DNA复制系统中重建染色体DNA复制，但非脊椎动物的Y RNA则不能。脊椎动物Y RNA双链茎中的一个保守核苷酸序列基序与Y RNA功能相关。对人类Y1 RNA突变体的功能筛选确定了这个保守基序是体外重建DNA复制的一个关键决定因素。包含该RNA基序的双链RNA寡核苷酸足以重建DNA复制，但相应的DNA或随机序列RNA寡核苷酸则不行。在完整细胞中，野生型hY1或保守的RNA双链体能够在针对hY3 RNA进行RNA干扰后挽救DNA复制的抑制。因此，我们鉴定出了一种在脊椎动物Y RNA进化中保守的新RNA基序，它对于人类染色体DNA复制中Y RNA的功能是必需且充分的。
