Structural basis for terminal loop recognition and processing of pri-miRNA-18a by hnRNP A1

Abstract

Running title: Terminal loop control of miRNA biogenesis not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/178855 doi: bioRxiv preprint first posted online Aug. 23, 2017; 2 Post-transcriptional mechanisms play a predominant role in the control of microRNA (miRNA) production. Recognition of the terminal loop of precursor miRNAs by RNA-binding proteins (RBPs) influences their processing; however, the mechanistic and structural basis for how levels of individual or subsets of miRNAs are regulated is mostly unexplored. We previously described a role for hnRNP A1, an RBP implicated in many aspects of RNA processing, as an auxiliary factor that promotes the Microprocessor-mediated processing of pri-mir-18a. Here, we reveal the mechanistic basis for this stimulatory role of hnRNP A1 by combining integrative structural biology with biochemical and functional assays. We demonstrate that hnRNP A1 forms a 1:1 complex with pri-mir-18a that involves binding of both RNA recognition motifs (RRMs) to cognate RNA sequence motifs in the conserved terminal loop of pri-mir-18a.Terminal loop binding induces an allosteric destabilization of base-pairing in the pri-mir-18a stem that promotes its down-stream processing. Our results highlight terminal loop RNA recognition by RNA-binding proteins as a general principle of miRNA biogenesis and regulation.Keywords: microRNA biogenesis; miR-18a, miR-17-92 cluster; hnRNP A1; nuclear magnetic resonance (NMR); X-ray crystallography; small angle X-ray/neutron scattering (SAXS/SANS); RNA recognition motif (RRM)not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/178855 doi: bioRxiv preprint first posted online Aug. 23, 2017; 3 MicroRNAs (miRNAs, miRs) are a class of highly conserved small non-coding RNAs that play a crucial role in the regulation of gene expression. They are involved in a variety of biological processes including cell growth, proliferation and differentiation 1 . Mature miRNAs are generated by two RNA cleavage steps involving nuclear and cytoplasmic RNase III enzymes (Drosha and Dicer, respectively). Primary miRNA (pri-miRNA or pri-mir) transcripts are cropped by the Microprocessor complex (comprising Drosha and DGCR8) in the nucleus forming ~70 nucleotide (nt) stem-loop precursor miRNAs (pre-miRNAs or pre-mir), which, following export to the cytoplasm, are further processed by Dicer into mature miRNAs (reviewed in 2 ). Many miRNA genes in higher organisms are transcribed together as a cluster 3 . A prototypical example is the miR-17-92 cluster that is encoded as an intronic polycistron on chromosome 13 in humans. This cluster encodes six individual miRNAs that are highly conserved in vertebrates (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, miR-92a-1, reviewed in 4 ). The miR-17-92 cluster is frequently amplified a...

Idiosyncrasies of hnRNP A1-RNA recognition: Can binding mode influence function

“…In this system, substitution of the adenosine with cytosine decreased UP1 binding affinity to SL3 ESS3 by ~10 fold [130] and the native adenosine showed a large NMR chemical shift perturbation upon complex formation [131]. A similar fold change in UP1 binding affinity for the 5′-AGUAGAUUAGCA-3′ 12mer, which mimics the apical loop of mir-18a, was also observed when either of the two internal AG dinucleotides was mutated to UU [132]. In this system, both RRMs of UP1 interact with the individual 5′-UAG-3′ elements as a 1:1 complex (more on this below).…”

“…Conversely, intact hnRNP A1 was shown to unwind a stem loop when given a running start by cooperatively spreading from a downstream 5′-UAGGGU-3′ sequence element [135]. NMR studies monitoring UP1 binding directly to stem loops shows the protein only locally perturbs RNA structure [131,132,136]. All this data shows the mechanisms by which hnRNP A1 interacts with cognate RNAs are context dependent.…”

“…In addition to the structures just described, a crystal structure of UP1 in complex with a single strand 12mer RNA (5′-AGUAGAUUAGCA-3′) that mimics that apical loop of mir-18a was also determined [132]. The UP1-12mer crystalized as a homodimer with 2:2 stoichiometry analogous to the UP1-DNA structure.…”

“…In some cases, the models were determined by integrating multiple data sets along with validation by mutagenesis. While each model offers intriguing perspectives into the mechanisms by which hnRNP A1 binds different RNA elements, they should be interpreted with caution given the nature by which they were determined [131,132,135,150].…”

“…The interaction of hnRNP A1 with primary mir-18a represents a more complex binding mode since its apical loop region contains dual 5′-UAG-3′ motifs separated by two spacer residues. A data-inspired model of the UP1-mir-18a complex was determined by integrating NMR (including PRE and RDC restraints), SAXS, mutagenesis and X-ray crystallography [132]. Despite having crystalized as a dimer with a 2:2 stoichiometry, the UP1-12mer structure provided information on the points of contact between the 5′-UAG-3′ motifs and the respective RRMs.…”