Abstract

Prior work demonstrates that mammalian microRNA (miRNA or miR) expression requires RNA polymerase II (Pol II). However, the transcriptional requirements of many miRNAs remain untested. Our genomic analysis of miRNAs in the human chromosome 19 miRNA cluster (C19MC) revealed that they are interspersed among Alu repeats. Because Alu transcription occurs through RNA Pol III recruitment, and we found that Alu elements upstream of C19MC miRNAs retain sequences important for Pol III activity, we tested the promoter requirements of C19MC miRNAs. Chromatin immunoprecipitation and cell-free transcription assays showed that Pol III, but not Pol II, is associated with miRNA genomic sequence and sufficient for transcription. Moreover, the mature miRNA sequences of approximately 50 additional human miRNAs lie within Alu and other known repetitive elements. These findings extend the current view of miRNA origins and the transcriptional machinery driving their expression. Over 450 miRNAs have been described in the human genome 1 . miRNAs are important in human development, oncogenesis and immunity, and they have drawn renewed attention to the small noncoding elements of the transcriptome. miRNAs are short (17-25 base pairs (bp)) noncoding RNAs that guide cellular machinery to specific messenger RNAs 2,3 to control expression. Initial miRNA transcripts can be several thousand base pairs in length, and they are processed to produce B70-bp stem-loops (pre-miRNAs) before nuclear export 4 . Upon entering the cytoplasm, the RNA-induced silencing complex (RISC) cleaves and denatures pre-miRNAs to produce the functionally mature, single-stranded miRNAs 4 . Through complementary base pairing to specific protein-coding mRNA transcripts, miRNAs direct mRNA silencing by a variety of mechanisms, including message degradation 5 , deadenylation 6 and translational repression 7 . The evolutionary conservation of miRNAs has been described 8 , as has their use of RNA polymerase II (Pol II) promoters for control of expression 9-11 . A recent study described approximately ten mammalian miRNAs created by a repetitive element transposition and subsequent transcription across two tandem, inverted repetitive elements 12 . In this study, we tested if other miRNAs possess sequences repetitive in origin, and the transcriptional requirements of a dense cluster of human miRNAs interspersed among repetitive Alu elements on chromosome 19. RESULTS miRNAs localize to repeats within the human genome We expanded prior studies by screening sequences flanking all known human miRNAs for repetitive elements (500 bp upstream and down stream) and annotating them using the Censor web server at the Genetic Information Research Institute 13 . Overall, we found that the mature sequences from B50 human miRNAs reside within repetitive elements Upstream Alus express downstream miRNAs We used degenerate primers and thermostable polymerase to first confirm that miRNAs within the C19MC are transcribed in vivo (Supplementary Table 2 online). To discern how the C19MC miRNAs are expressed, we used one of these miRNAs, miR-517a, and sequences immediately upstream, which consist of an Alu We next made reporter constructs to test whether the hairpins expressed from the miR-517a 5¢ Alu had silencing activity. These reporters (517-AS and LacZ-AS) produced Renilla luciferase transcripts with target sequences for miR-517a or shLacZ cloned into the 3¢ UTR