The STAR protein QKI-7 recruits PAPD4 to regulate post-transcriptional polyadenylation of target mRNAs

Abstract

Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27kip1 and β-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27kip1 mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells.

TDP‐43 accelerates deadenylation of target mRNAs by recruiting Caf1 deadenylase

“…We therefore confirmed the effect of TDP‐43 using a tethering strategy in which TDP‐43 was fused to the coat protein of bacteriophage MS2 and the MS2 binding site was incorporated into a reporter mRNA (Fig. A) . HEK293T cells were cotransfected with three plasmids: the first carried a Flag‐tagged β‐globin reporter mini gene (BGG(1–39)) that produces mRNA with appended MS2‐binding sites in its 3′‐UTR (FBG(1–39)‐MS2bs), the second is a reference expressing 5xFlag‐EGFP‐MALAT1 as transfection/loading control, and the third expresses either 5xMyc‐tagged TDP‐43 or GST fused to the MS2 coat protein coding sequence.…”

ABCE1 Acts as a Positive Regulator of Exogenous RNA Decay

“…5×Flag-EGFP mRNA was transcribed as described previously [5]. p5×Flag-RNase L, p5×Myc-RNase L, p5×Flag-Pelota and p5×Myc-GST were previously described [5,21]. 2-5A, a tetra-adenylate (pA2 p5 A2 p5 A2 p5 A), was synthesized, purified by reversed phase HPLC and analyzed by MALDI-TOF/MS as described previously [22].…”

Direct evidence that Ataxin-2 is a translational activator mediating cytoplasmic polyadenylation

“…These interactions are mediated by the C-terminal PABC domain of PABPC1 and PABPC1-interacting motif 2 (PAM2) of eRF3, Pan3 and Tob (10,12,15,16). (35). Figure 1.…”