Abstract:
The human U1 snRNA is encoded by a multigene family consisting of transcribed variants and defective pseudogenes. Many variant U1 (vU1) snRNAs have been demonstrated to not only be transcribed but also processed by the addition of a trimethylated guanosine cap, packaged into snRNPs, and assembled into spliceosomes; however, their capacity to facilitate pre-mRNA splicing has, so far, not been tested. A recent systematic analysis of the human snRNA genes identified 178 U1 snRNA genes that are present in the genome as either tandem arrays or single genes on multiple chromosomes. Of these, 15 were found to be expressed in human tissues and cell lines, although at significantly low levels from their endogenous loci, <0.001% of the canonical U1 snRNA. In this study, we found that placing the variants in the context of the regulatory elements of the RNU1-1 gene improves the expression of many variants to levels comparable to the canonical U1 snRNA. Application of a previously established HeLa cell-based minigene reporter assay to examine the capacity of the vU1 snRNAs to support pre-mRNA splicing revealed that even though the exogenously expressed variant snRNAs were enriched in the nucleus, only a few had a measurable effect on splicing.
摘要:
人UlsnRNA由多基因家族编码,所述多基因家族由转录的变体和缺陷的假基因组成。已经证明许多变体U1(vU1)snRNA不仅可以转录,而且可以通过添加三甲基化鸟苷帽进行处理,打包到snRNPs中,组装成剪接体,然而,它们促进前mRNA剪接的能力,到目前为止,没有经过测试。最近对人类snRNA基因的系统分析鉴定了178个U1snRNA基因,这些基因在基因组中以串联阵列或多个染色体上的单个基因存在。其中,发现15在人体组织和细胞系中表达,尽管其内源性基因座的水平明显较低,小于规范U1snRNA的0.001%。在这项研究中,我们发现,将变体置于RNA1-1基因调控元件的背景下,可将许多变体的表达提高到与标准U1snRNA相当的水平.应用先前建立的基于HeLa细胞的小基因报告基因测定来检查vU1snRNA支持pre-mRNA剪接的能力表明,即使外源表达的变体snRNA在细胞核中富集,只有少数对剪接有可测量的影响。