DGCR8 HITS-CLIP reveals novel functions for the Microprocessor (CLIP-seq)

The Drosha-DGCR8 complex (Microprocessor) is required for microRNA (miRNA) biogenesis. DGCR8 contains two double-stranded RNA binding motifs that recognize the RNA substrate, whereas Drosha functions as the endonuclease. We have used high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify endogenous RNA targets of DGCR8 in mammalian cells. Unexpectedly, miRNAs were not the most abundant targets. DGCR8-bound RNAs comprised several hundred mRNAs as well as snoRNAs and long non-coding RNAs. We found that DGCR8 together with Drosha controls the abundance of several mRNAs, as well as long non-coding RNAs, such as MALAT-1. By contrast, the DGCR8-mediated cleavage of snoRNAs is independent of Drosha, suggesting the involvement of DGCR8 in cellular complexes with other endonucleases. Interestingly, binding of DGCR8 to cassette exons, acts as a novel mechanism to regulate the relative abundance of alternatively spliced isoforms. Collectively, these data provide new insights in the complex role of DGCR8 in controlling the fate of several classes of RNAs. Comparison of RNAs associated to both endogenous (D8) and overexpressed (T7) DGCR8 in HEK293T cells

Drosha-DGCR8复合物（Microprocessor）是微小RNA（microRNA, miRNA）生物发生过程所必需的。DGCR8含有两个可识别RNA底物的双链RNA结合基序（double-stranded RNA binding motifs），而Drosha则作为核酸内切酶发挥功能。本研究采用紫外交联免疫沉淀结合高通量测序（HITS-CLIP）技术，分离RNA并进行高通量测序，以此鉴定哺乳动物细胞内DGCR8的内源性RNA靶标。出乎意料的是，miRNA并非丰度最高的靶标。DGCR8结合的RNA涵盖数百条信使RNA（mRNA）、核仁小RNA（snoRNAs）以及长链非编码RNA（long non-coding RNAs）。研究发现，DGCR8可与Drosha共同调控多条mRNA及诸如MALAT-1这类长链非编码RNA的丰度。与之形成对照的是，DGCR8介导的核仁小RNA切割过程并不依赖Drosha，这提示DGCR8可与其他核酸内切酶共同参与细胞复合物的形成。值得注意的是，DGCR8结合盒式外显子（cassette exons）这一作用，可作为一种全新的调控可变剪接异构体相对丰度的机制。综上，本研究数据为DGCR8在调控多类RNA命运过程中的复杂作用提供了全新的研究视角。同时，本研究还对HEK293T细胞中内源性（D8）与过表达（T7）的DGCR8结合RNA开展了对比分析。