AUF1 promotes let-7b loading on Argonaute 2

Abstract

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 micro-RNA let-7b in vitro (K d = ∼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.

AUF1 regulation of coding and noncoding RNA

“…PAR‐CLIP analyses from human embryonic kidney cells identified 14 distinct miRNA targets of AUF1 in this cell model, including 4 members of the let‐7 family (let‐7a‐2, ‐7b, ‐7f‐2, and ‐7i) . Subsequent biochemical assays verified that all AUF1 isoforms directly interacted with let‐7b and miR‐21, both of which include UU‐ and UG‐rich sequences, and in the case of let‐7b demonstrated surprisingly strong affinity given the short length (22 nt) of the miRNA substrate (described above). Several functional consequences could be envisioned for RBP binding to specific miRNAs, including sequestering them from assembly into miRISCs or altering miRNA turnover kinetics.…”

“…However, in the case of AUF1, focusing principally on binding to let‐7b, a different role was identified. Silencing AUF1 expression in cells had no effect on levels of let‐7b or other tested miRNAs, indicating that AUF1 binding was unlikely to impact their turnover. However, in the absence of AUF1, let‐7b loading onto AGO2 was dramatically inhibited (Figure , arrow b), which in turn abrogated its ability to accelerate decay of let‐7b‐targeted mRNAs.…”

“…However, a paradoxical result from the PAR‐CLIP screens was the identification of several miRNA partners for AUF1 which, measuring as small as 20 nt in length, are far shorter than the minimal ARE‐based substrates required for high‐affinity RNP formation. Subsequent experiments revealed that one GU‐rich miRNA, let‐7b, bound p37 AUF1 directly in vitro with a K d of 6 nM, far superior to the K d = 26 nM observed for p37 AUF1 binding to a comparably sized AU‐rich RNA substrate . How AUF1 is able to form such stable RNP complexes with let‐7b and possibly other miRNA targets is unknown, but it is likely that distinct sequence and/or structural determinants within these substrates present optimal contacts for protein recognition.…”

Profiling of m6A RNA modifications identified an age‐associated regulation of AGO2 mRNA stability

“…Although a subset of let‐7 family miRNAs are downregulated, the rest are not affected in their abundance. Differential expression of let‐7 family miRNAs may arise from the existence of novel‐miRNA‐binding proteins such as AUF1 and HuR possibly affecting the stability of mature miRNAs (Yoon, Abdelmohsen, Kim, et al., 2013; Yoon et al., 2015). Thus, miRNA abundance could be influenced by other RNA‐binding proteins whose expression and/or activity depends on a cellular context.…”

Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity

“…AUF1 has been shown to shuttle between the nucleus and the cytoplasm; the cytoplasm being where it promotes ARE-mRNA decay. At steady-state AUF1 is primarily nuclear with export to the cytoplasm occurring as a result of specific mRNA association for decay (Moore et al, 2014; Sarkar et al, 2003a; Suzuki et al, 2005; Yoon et al, 2015; He and Schneider, 2006). Collectively, these data demonstrate that AUF1 is only expressed in activated satellite cells in skeletal muscle, and not in muscle fibers.…”