In vivo PAR-CLIP (viP-CLIP) of liver TIAL1 unveils targets regulating cholesterol synthesis and secretion

Abstract

System-wide cross-linking and immunoprecipitation (CLIP) approaches have unveiled regulatory mechanisms of RNA-binding proteins (RBPs) mainly in cultured cells due to limitations in the cross-linking efficiency of tissues. Here, we describe viP-CLIP (in vivo PAR-CLIP), a method capable of identifying RBP targets in mammalian tissues, thereby facilitating the functional analysis of RBP-regulatory networks in vivo. We applied viP-CLIP to mouse livers and identified Insig2 and ApoB as prominent TIAL1 target transcripts, indicating an important role of TIAL1 in cholesterol synthesis and secretion. The functional relevance of these targets was confirmed by showing that TIAL1 influences their translation in hepatocytes. Mutant Tial1 mice exhibit altered cholesterol synthesis, APOB secretion and plasma cholesterol levels. Our results demonstrate that viP-CLIP can identify physiologically relevant RBP targets by finding a factor implicated in the negative feedback regulation of cholesterol biosynthesis.

VLDL Biogenesis and Secretion: It Takes a Village

“…This review integrates the new insights from the recent literature with what had been learned previously into an extended, detailed working model of VLDL biogenesis and secretion (Figure 2). It has been well-established that VLDL is regulated at the posttranslational level (reviewed in the study by Ginsberg and Fisher 86 ), but we here list several studies that have provided evidence for additional regulation at the transcriptional level (HUR 13 ) and strong evidence for regulation at the translational level at the outer ER membrane (VIGILIN and TIAL1 14,15 ). In addition, LZP 19 and MDM2 20 can be added as members of a large number of proteins that were previously shown to affect apoB/VLDL at the early posttranslational level (Table ).…”

“…The same investigators recently showed that TIAL1 (TIA1 cytotoxic granule-associated RNA-binding protein like 1) 14 -another RNA-binding protein-could affect VLDL metabolism. Loss of TIAL1 in mice markedly reduces apoB synthesis by 50% with a 31% reduction in VLDL secretion but increases in hepatic triglycerides and cholesterol.…”

“…Loss of TIAL1 in mice markedly reduces apoB synthesis by 50% with a 31% reduction in VLDL secretion but increases in hepatic triglycerides and cholesterol. 14 The authors suggest that TIAL1 may recruit apoB to translationally active polysomes or prevent translational repressors of other RNA-binding proteins or microRNAs.…”

“…ApoB-the core protein of VLDL-is widely accepted to be produced constitutively. There is, however, increasing evidence that gene expression is also regulated at the mRNA level through, for example, HUR (human antigen R 13 ), TIAL1 (TIA1 cytotoxic granule-associated RNA-binding protein like 1), 14 VIGILIN, 15 and miRNAs. 16 In the nucleus, we have depicted the APOBEC1 (Apob100 mRNA editing enzyme catalytic polypeptide 1) to make clear that in mouse hepatocytes but not human hepatocytes, APOBEC1 generates apoB48 mRNA, which encodes for a truncated (48%) portion of the full-length APOB100 protein.…”