The Human CCHC-type Zinc Finger Nucleic Acid-Binding Protein Binds G-Rich Elements in Target mRNA Coding Sequences and Promotes Translation

Abstract

Summary The CCHC-type Zinc Finger Nucleic Acid Binding Protein (CNBP/ZNF9) is conserved in eukaryotes and essential for embryonic development in mammals. It has been implicated in transcriptional as well as post-transcriptional gene regulation; however, its nucleic acid ligands and molecular function remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and function. We used Photoactivatable Ribonucleoside Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) to identify 8420 CNBP binding sites on 4178 mRNAs. CNBP preferentially bound G-rich elements in the target mRNA coding sequences, most of which were previously found to form G-quadruplex and other stable structures in vitro. Functional analyses, including RNA sequencing, ribosome profiling, and quantitative mass spectrometry, revealed that CNBP binding did not influence target mRNA abundance but rather increased their translational efficiency. Considering that CNBP binding prevented G quadruplex structure formation in vitro, we hypothesize that CNBP is supporting translation by resolving stable structures on mRNAs.

Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them

“…FMRP might inhibit translation initiation or elongation by binding to and stabilizing RG4 structures and recruiting trans- acting factors or by direct binding and stalling of the translating ribosome 79,80 . Other examples exist of RBPs that promote translation by destabilizing RG4 structures in the CDS 81 , further highlighting the diverse roles of stable RNA tertiary structures in translation regulation.…”

RNA G-Quadruplexes in Biology: Principles and Molecular Mechanisms

“…A recent paper by Benhalevy et al . suggests that CNBP/ZNF9 is one of such proteins that binds G-rich elements in mRNAs, most of which form G4s in vitro 92 . Functionally, CNBP increases target mRNA translation efficiency by resolving G4s and other stable structures on mRNAs.…”

G-quadruplex formation on specific surface-exposed regions of the human ribosomal RNA

“…The biotinylation of GQES7-1 did not disrupt the G-quadruplexes (Figure S.5). Several known G-quadruplex-binding proteins were pulled down by this assay (CNBP, YBOX1, hnRP F, hnRP H, DDX21, DDX17) [36][37][38][39][40][41] . Also, a significant number of helicases were identified (DDX3, CNBP, DDX21, DDX17).…”

“…Also, a significant number of helicases were identified (DDX3, CNBP, DDX21, DDX17). All these helicases except DDX3 have been reported to unfold Gquadruplexes 36,[40][41] . In addition, a significant number of heterogeneous nuclear ribonucleoproteins (hnRNPs) were bound to GQES7-1, including hnRNP G-T/RMXL2, hnRNP M, hnRNP G/RBMX, hnRNP H2, hnRNP H, hnRNP F, hnRNP H3, and FUS.…”