A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis

Abstract

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.

LIN28 Selectively Modulates a Subclass of Let-7 MicroRNAs

“…To further validate our hypothesis and exclude the possibility that the selective binding observed from CLIP data is due to a technical bias (e.g., differences in crosslinking efficiency), we examined a recently published dataset of pri-miRNA-binding interactomes in 11 cell lines, in which pri-miRNA-hairpin-interacting proteins were captured using an RNA-mediated protein pull-down assay followed by mass spectrometry analysis (Treiber et al, 2017). A number of miRNA precursors including all members of the let-7 family were used as a bait to identify specific protein interactors.…”

“…Both LIN28A and LIN28B showed a greater preference for binding of CSD + pri-let-7 hairpins (P=0.0008 and 0.005, respectively, t-test; Figure 2D). The spectrum counts from the mass spectrometry data is not quantitative by nature, although a normalization procedure was performed to allow unbiased comparison of protein pull-down using different pri-miRNA-hairpin baits (Treiber et al, 2017). To obtain a more quantitative measure of the interaction between LIN28 and the two subclasses of pre-let-7 miRNAs, we performed the similar RNA-mediated protein pull-down assay using all 12 let-7 precursors and endogenous LIN28 from NTera2 teratocarcinoma cell line under harsh washing conditions; the pri-miR-18b hairpin without LIN28 binding motifs was used as a negative control.…”

“…The initial analysis of LIN28 pull-down using let-7 pri-miRNA haipin baits was performed using published interactome capture data in 11 different cell lines (Treiber et al, 2017). Statistical analysis of LIN28A and LIN28B pull down using CSD + and CSD − let-7 precursors was performed using normalized mass spectrometry-based spectrum counts derived by the original study, which were the percentage of total counts, averaged over all cell lines in which the protein was identified.…”

Impact of RNA–Protein Interaction Modes on Translation Control: The Versatile Multidomain Protein Gemin5

“…In addition to short sequence motifs, the RNA secondary structure and sequence context contribute to the RNA–protein interaction specificity (Figure a). Accordingly, proteins with different RBDs can exhibit different RNA‐binding properties, as shown by proteins regulating microRNA biogenesis . For example, ZF‐containing RBPs, such as transcription factor IIIA (TFIIIA) or muscleblind‐like1 (MBNL1), favor binding to structured motifs.…”

MicroRNAs in Acute Kidney Injury

“…In excess of 2,500 microRNAs have now been identified in humans [4]. Typically microRNAs bind to mRNA at the 3′ untranslated region in a sequence specific fashion and, once bound, silencing is achieved through a combination of direct inhibition of ribosomal translation and acceleration of mRNA degradation [3].…”