Beyond Secondary Structure: Primary-Sequence Determinants License Pri-miRNA Hairpins for Processing

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Cell (Impact Factor: 32.24). 02/2013; 152(4):844-58. DOI: 10.1016/j.cell.2013.01.031
Source: PubMed


To use microRNAs to downregulate mRNA targets, cells must first process these ∼22 nt RNAs from primary transcripts (pri-miRNAs). These transcripts form RNA hairpins important for processing, but additional determinants must distinguish pri-miRNAs from the many other hairpin-containing transcripts expressed in each cell. Illustrating the complexity of this recognition, we show that most Caenorhabditis elegans pri-miRNAs lack determinants required for processing in human cells. To find these determinants, we generated many variants of four human pri-miRNAs, sequenced millions that retained function, and compared them with the starting variants. Our results confirmed the importance of pairing in the stem and revealed three primary-sequence determinants, including an SRp20-binding motif (CNNC) found downstream of most pri-miRNA hairpins in bilaterian animals, but not in nematodes. Adding this and other determinants to C. elegans pri-miRNAs imparted efficient processing in human cells, thereby confirming the importance of primary-sequence determinants for distinguishing pri-miRNAs from other hairpin-containing transcripts.

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    • "However, only one strand of the duplex is retained in the RISC (the " guide " miRNA), while the other is discarded and degraded, resulting in a strong bias for guide strands in the miRNA pool (Khvorova et al., 2003; Schwarz et al., 2003). This miRNA maturation process is mainly regulated at the level of nuclear processing, and sequence determinants and auxiliary factors also contribute to its regulation (Auyeung et al., 2013; Mori et al., 2014). There is evidence that a small proportion of miRNAs (<1%) are produced by non-canonical mechanisms, including: Droshaindependent mechanisms (as in the case of " mirtrons " ), in which mRNA splicing produces a small RNA hairpin thereby bypassing Drosha processing (Berezikov et al., 2007); and Dicer-independent mechanisms (as in the case of miR-451), in which a short stemloop is not loaded onto Dicer but directly processed by Ago2 (Yang et al., 2010). "
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    • "One of the most significantly enriched sequence motifs that we find in flanking regions of efficiently cleaved pre-miRNAs is the downstream CNNC motif that has been derived by Auyeung et al. (2013) from a large-scale in vitro screen using variants of four model pri-miRNA transcripts and was shown to bind SRP20, underlining the potential importance of this motif for pri-miRNA processing both in vitro and in vivo. Also, the DEAD box helicase p72 has been found to bind a related VCAUCH motif and affect the processing of pri-miRNAs (Mori et al., 2014). "
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    ABSTRACT: In miRNA biogenesis, pri-miRNA transcripts are converted into pre-miRNA hairpins. The in vivo properties of this process remain enigmatic. Here, we determine in vivo transcriptome-wide pri-miRNA processing using next-generation sequencing of chromatin-associated pri-miRNAs. We identify a distinctive Microprocessor signature in the transcriptome profile from which efficiency of the endogenous processing event can be accurately quantified. This analysis reveals differential susceptibility to Microprocessor cleavage as a key regulatory step in miRNA biogenesis. Processing is highly variable among pri-miRNAs and a better predictor of miRNA abundance than primary transcription itself. Processing is also largely stable across three cell lines, suggesting a major contribution of sequence determinants. On the basis of differential processing efficiencies, we define functionality for short sequence features adjacent to the pre-miRNA hairpin. In conclusion, we identify Microprocessor as the main hub for diversified miRNA output and suggest a role for uncoupling miRNA biogenesis from host gene expression.
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    • "These findings extend our previous inference that a limited number of the hundreds of thousands of plausible miRNA-like hairpins are actually competent for biogenesis into specific small RNAs (Berezikov et al. 2010, 2011). Recent studies of highly complex pools of pri-miRNA hairpins reveal additional determinants of successful Drosha substrates (Auyeung et al. 2013), and presumably other features of ''successful'' miRNA hairpins remain to be elucidated. On the other hand, these extensive cell line data allowed us to uncover a diversity of novel endo-siRNA and piRNA loci. "
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