AUF1 promotes let-7b loading on Argonaute 2.

Eukaryotic gene expression is tightly regulated post-transcriptionally by RNA-binding proteins (RBPs) and microRNAs. The RBP AU-rich-binding factor 1 (AUF1) isoform p37 was found to have high affinity for the microRNA let-7b in vitro (Kd = ∼ 6 nM) in cells. Ribonucleoprotein immunoprecipitation, in vitro association, and single-molecule-binding analyses revealed that AUF1 promoted let-7b loading onto Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex (RISC). In turn, AGO2-let-7 triggered target mRNA decay. Our findings uncover a novel mechanism by which AUF1 binding and transfer of microRNA let-7 to AGO2 facilitates let-7-elicited gene silencing.

Hsp70's RNA-binding and mRNA-stabilizing activities are independent of its protein chaperone functions.

Hsp70 is a protein chaperone that prevents protein aggregation and aids protein folding by binding to hydrophobic peptide domains through a reversible mechanism directed by an ATPase cycle. However, Hsp70 also binds U-rich RNA including some AU-rich elements (AREs) that regulate the decay kinetics of select mRNAs and has recently been shown to bind and stabilize some ARE-containing transcripts in cells. Previous studies indicated that both the ATP- and peptide-binding domains of Hsp70 contributed to the stability of Hsp70-RNA complexes and that ATP might inhibit RNA recruitment. This suggested the possibility that RNA binding by Hsp70 might mimic features of its peptide-directed chaperone activities. Here, using purified, cofactor-free preparations of recombinant human Hsp70 and quantitative biochemical approaches, we found that high-affinity RNA binding requires at least 30 nucleotides of RNA sequence but is independent of Hsp70's nucleotide-bound status, ATPase activity, or peptide-binding roles. Furthermore, although both the ATP- and peptide-binding domains of Hsp70 could form complexes with an ARE sequence from VEGFA mRNA in vitro, only the peptide-binding domain could recover cellular VEGFA mRNA in ribonucleoprotein immunoprecipitations. Finally, Hsp70-directed stabilization of VEGFA mRNA in cells was mediated exclusively by the protein's peptide-binding domain. Together, these findings indicate that the RNA-binding and mRNA-stabilizing functions of Hsp70 are independent of its protein chaperone cycle but also provide potential mechanical explanations for several well-established and recently discovered cytoprotective and RNA-based Hsp70 functions.

Post-transcriptional control of gene expression by AUF1: mechanisms, physiological targets, and regulation.

AUF1 is a family of four proteins generated by alternative pre-mRNA splicing that form high affinity complexes with AU-rich, mRNA-destabilizing sequences located within the 3' untranslated regions of many labile mRNAs. While AUF1 binding is most frequently associated with accelerated mRNA decay, emerging examples have demonstrated roles as a mRNA stabilizer or even translational regulator for specific transcripts. In this review, we summarize recent advances in our understanding of mRNA recognition by AUF1 and the biochemical and functional consequences of these interactions. In addition, unique properties of individual AUF1 isoforms and the roles of these proteins in modulating expression of genes associated with inflammatory, neoplastic, and cardiac diseases are discussed. Finally, we describe mechanisms that regulate AUF1 expression in cells, and current knowledge of regulatory switches that modulate the cellular levels and/or activities of AUF1 isoforms through distinct protein post-translational modifications. This article is part of a Special Issue entitled: RNA Decay mechanisms.

AUF1 regulation of coding and noncoding RNA.

AUF1 is a family of four RNA-binding proteins (RBPs) generated by alternative pre-messenger RNA (pre-mRNA) splicing, with canonical roles in controlling the stability and/or translation of mRNA targets based on recognition of AU-rich sequences within mRNA 3' untranslated regions. However, recent studies identifying AUF1 target sites across the transcriptome have revealed that these canonical functions are but a subset of its roles in posttranscriptional regulation of gene expression. In this review, we describe recent developments in our understanding of the RNA-binding properties of AUF1 together with their biochemical implications and roles in directing mRNA decay and translation. This is then followed by a survey of newly discovered activities for AUF1 proteins in control of miRNA synthesis and function, including miRNA assembly into microRNA (miRNA)-loaded RNA-induced silencing complexes (miRISCs), miRISC targeting to mRNA substrates, interplay with an expanding network of other cellular RBPs, and reciprocal regulatory relationships between miRNA and AUF1 synthesis. Finally, we discuss recently reported relationships between AUF1 and long noncoding RNAs and regulatory roles on viral RNA substrates. Cumulatively, these findings have significantly expanded our appreciation of the scope and diversity of AUF1 functions in the cell, and are prompting an exciting array of new questions moving forward. WIREs RNA 2017, 8:e1393. doi: 10.1002/wrna.1393 For further resources related to this article, please visit the WIREs website.

PAR-CLIP analysis uncovers AUF1 impact on target RNA fate and genome integrity.

Post-transcriptional gene regulation is robustly regulated by RNA-binding proteins (RBPs). Here we describe the collection of RNAs regulated by AUF1 (AU-binding factor 1), an RBP linked to cancer, inflammation and aging. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis reveals that AUF1 primarily recognizes U-/GU-rich sequences in mRNAs and noncoding RNAs and influences target transcript fate in three main directions. First, AUF1 lowers the steady-state levels of numerous target RNAs, including long noncoding RNA NEAT1, in turn affecting the organization of nuclear paraspeckles. Second, AUF1 does not change the abundance of many target RNAs, but ribosome profiling reveals that AUF1 promotes the translation of numerous mRNAs in this group. Third, AUF1 unexpectedly enhances the steady-state levels of several target mRNAs encoding DNA-maintenance proteins. Through its actions on target RNAs, AUF1 preserves genomic integrity, in agreement with the AUF1-elicited prevention of premature cellular senescence.