RNA binding protein PRRC2B mediates translation of specific proteins and regulates cell cycle progression [PAR_Clip-seq]

Accumulating evidence suggests that posttranscriptional regulation of gene expression, including regulation of RNA splicing, transport, modification, translation, and degradation, primarily relies on RNA binding proteins (RBPs). However, the functions of many RBPs remain understudied. Here, we characterized the function of a novel RBP, Proline-Rich Coiled-coil 2B (PRRC2B). Through photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and deep sequencing, we identified transcriptome-wide CU- or GA-rich PRRC2B binding sites around the translation initiation codon on a specific cohort of mRNAs in HEK293T cells. These PRRC2B target mRNAs, including oncogenes and cell cycle regulators such as CCND2 (cyclin D2), exhibited decreased translation upon PRRC2B knockdown as revealed by sequencing polysome-associated RNA, resulting in decreased G1/S phase transition and cell proliferation. Antisense oligonucleotides (ASOs) blocking PRRC2B-CCND2 mRNA interaction decreased CCND2 translation, thus inhibited G1/S transition and cell proliferation. Mechanistically, PRRC2B interactome capture analysis revealed RNA-independent interactions with eukaryotic initiation factors eIF4G2, eIF3, and FXR1. The interaction with eIF4G2 is essential for PRRC2B function since unlike wildtype PRRC2B, eIF4G2-interacting defective mutants failed to rescue the translation deficiency caused by PRRC2B knockdown. Taken together, our findings reveal that PRRC2B, by interacting with eIF4G2, is essential for efficient translation of specific proteins required for cell cycle progression and cell proliferation. We performed PAR-CLIP-seq on HEK293T cells overexpressing Flag-tagged full-length and truncated PRRC2B. PRRC2B-bound RNAs were digested by RNase T1 to 19 - 35 nt long and extracted from immunoprecipitated PRRC2B-RNA complexes. Biological replicates were performed. The samples were subjected to libraries construction using the NEBNext® Small RNA Library Prep Set following the manufacturer’s instructions. Sequencing was performed on Illumina HiSeq2500 sequencer with 75 bp single-end sequencing in the Genomic Research Center from University of Rochester Medical Center.