Feed-Forward Microprocessing and Splicing Activities at a MicroRNA–Containing Intron

Maja M Janas,David E Fisher,David Golan,Carmit Levy,Rosa A Veguilla,Steffen Schubert,Noam Shomron,Carl D Novina,Mehdi Khaled,Jacob G Bernstein,Michael T Mcmanus

doi:10.1371/journal.pgen.1002330

Maja M Janas, David E Fisher + Show 9 more

Open Access

https://doi.org/10.1371/journal.pgen.1002330

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Abstract

The majority of mammalian microRNA (miRNA) genes reside within introns of protein-encoding and non-coding genes, yet the mechanisms coordinating primary transcript processing into both mature miRNA and spliced mRNA are poorly understood. Analysis of melanoma invasion suppressor miR-211 expressed from intron 6 of melastatin revealed that microprocessing of miR-211 promotes splicing of the exon 6–exon 7 junction of melastatin by a mechanism requiring the RNase III activity of Drosha. Additionally, mutations in the 5′ splice site (5′SS), but not in the 3′SS, branch point, or polypyrimidine tract of intron 6 reduced miR-211 biogenesis and Drosha recruitment to intron 6, indicating that 5′SS recognition by the spliceosome promotes microprocessing of miR-211. Globally, knockdown of U1 splicing factors reduced intronic miRNA expression. Our data demonstrate novel mutually-cooperative microprocessing and splicing activities at an intronic miRNA locus and suggest that the initiation of spliceosome assembly may promote microprocessing of intronic miRNAs.

Highlights

Most eukaryotic primary transcripts undergo nuclear splicing, which removes introns and joins exons in a process catalyzed by a multi-megadalton complex called the spliceosome
Our analysis of miR-211 biogenesis from intron 6 of MicroRNA genes are transcribed as long primary RNAs containing local hairpins that are excised by the Microprocessor complex minimally composed of Drosha and DGCR8
We recently reported that in melanoma, a miRNA expressed from intron 6 of melastatin assumes the tumor suppressive function of its host gene

Summary

Introduction

Most eukaryotic primary transcripts undergo nuclear splicing, which removes introns and joins exons in a process catalyzed by a multi-megadalton complex called the spliceosome. MiRNA-containing hairpins are cropped from primary miRNA transcripts (pri-miRNAs) by the Microprocessor, a protein complex minimally containing the nuclear RNase III enzyme Drosha and DGCR8 [4,5,6,7]. The melanocyte-specific gene melastatin and its hosted miR211 gene located in intron 6 are robustly reduced in invasive human melanomas [16,17]. We detected increased formation of exon 6-exon 7 junction relative to other melastatin exon-exon junctions which lack intronic miRNAs. Here we demonstrate that microprocessing of miR-211 promotes splicing of the exon 6-exon 7 junction of melastatin, that knockdown of Drosha and its binding partner DGCR8 reduces exon 6-exon 7 junction formation, and that the RNase III activity of Drosha is required to promote exon 6exon 7 junction formation.

Author Summary

Materials and Methods