MicroRNAs (miRNAs) are short non-coding RNAs that inhibit the expression of target genes by directly binding to their mRNAs. miRNAs are transcribed as precursor molecules, which are subsequently cleaved by the endoribonucleases Drosha and Dicer. Mature miRNAs are bound by a member of the Argonaute (AGO) protein family to form the RNA-induced silencing complex (RISC) in a process termed RISC loading. Advances in structural analyses of Drosha and Dicer complexes enabled elucidation of the mechanisms that drive these molecular machines. Transcription of miRNAs, their processing by Drosha and Dicer and RISC loading are key steps in miRNA biogenesis, and various additional factors facilitate, support or inhibit these processes. Recent work has revealed that regulatory factors not only coordinate individual miRNA processing steps but also connect miRNA biogenesis with other cellular processes. Protein phosphorylation, for example, links miRNA biogenesis to various signalling pathways, and such modifications are often associated with disease. Furthermore, not all miRNAs follow canonical processing routes, and many non-canonical miRNA biogenesis pathways have recently been characterized.

-methyladenine (mA) is found on many eukaryotic RNAs including mRNAs. mA modification has been implicated in mRNA stability and turnover, localization, or translation efficiency. A heterodimeric enzyme complex composed of METTL3 and METTL14 generates mA on mRNAs. METTL3/14 is found in the nucleus where it is localized to nuclear speckles and the splicing regulator WTAP is required for this distinct nuclear localization pattern. Although recent crystal structures revealed how the catalytic MT-A70 domains of METTL3 and METTL14 interact with each other, a more global architecture including WTAP and RNA interactions has not been reported so far. Here, we used recombinant proteins and mapped binding surfaces within the METTL3/14-WTAP complex. Furthermore, we identify nuclear localization signals and identify phosphorylation sites on the endogenous proteins. Using an in vitro methylation assay, we confirm that monomeric METTL3 is soluble and inactive while the catalytic center of METTL14 is degenerated and thus also inactive. In addition, we show that the C-terminal RGG repeats of METTL14 are required for METTL3/14 activity by contributing to RNA substrate binding. Our biochemical work identifies characteristic features of METTL3/14-WTAP and reveals novel insight into the overall architecture of this important enzyme complex.

During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.

TRIM-NHL proteins are conserved among metazoans and control cell fate decisions in various stem cell linages. The Drosophila TRIM-NHL protein Brain tumor (Brat) directs differentiation of neuronal stem cells by suppressing self-renewal factors. Brat is an RNA-binding protein and functions as a translational repressor. However, it is unknown which RNAs Brat regulates and how RNA-binding specificity is achieved. Using RNA immunoprecipitation and RNAcompete, we identify Brat-bound mRNAs in Drosophila embryos and define consensus binding motifs for Brat as well as a number of additional TRIM-NHL proteins, indicating that TRIM-NHL proteins are conserved, sequence-specific RNA-binding proteins. We demonstrate that Brat-mediated repression and direct RNA-binding depend on the identified motif and show that binding of the localization factor Miranda to the Brat-NHL domain inhibits Brat activity. Finally, to unravel the sequence specificity of the NHL domain, we crystallize the Brat-NHL domain in complex with RNA and present a high-resolution protein-RNA structure of this fold.

SummaryMicroRNAs (miRNAs) are considered as key regulators of literally all cellular pathways. Therefore, miRNA biosynthesis and their individual cellular functions must be tightly regulated as well. MiRNAs are transcribed as primary transcripts, which are processed to mature miRNAs in two consecutive maturation steps. Finally, the mature miRNA is incorporated into a miRNA-protein complex, where it directly interacts with a member of the Argonaute (Ago) protein family. The miRNA guides such protein complexes to partial complementary target sites, which are typically located in the 3' untranslated region (UTR) of mRNAs leading to inhibition of gene expression. MiRNA activity and abundance is regulated on various levels ranging from transcription and processing to target site binding and miRNA stability. Recent advances in our understanding of how miRNA activity is regulated in mammalian cells are summarised and discussed in this review article.