Identification and Characterization of Proteins That Selectively Interact with Isoforms of the mRNA Binding Protein AUF1 (hnRNP D)

Abstract

The mRNAs that encode certain cytokines and proto-oncogenes frequently contain a typical AU-rich motif that is located in their 3'-untranslated region. The protein AUF1 is the first factor identified that binds to AU-rich regions and mediates the fast degradation of the target mRNAs. AUF1 exists as four different isoforms (p37, p40, p42 and p45) that are generated by alternative splicing. The fact that AUF1 does not degrade mRNA itself had led to the suggestion that other AUF1 interacting proteins might be involved in the process of selective mRNA degradation. Here we used the yeast two-hybrid system in order to identify proteins that bind to AUF1. We detected AUF1 itself, as well as the ubiquitin-conjugating enzyme E2I and three RNA binding proteins: NSEP-1, NSAP-1 and IMP-2, as AUF1 interacting proteins. We confirmed all interactions in vitro and mapped the protein domains that are involved in the interaction with AUF1. Gel-shift assays with the recombinant purified proteins suggest that the interacting proteins and AUF1 can bind simultaneously to an AU-rich RNA oligonucleotide. Most interestingly, the AUF1 interacting protein NSEP-1 showed an endoribonuclease activity in vitro. These data suggest the possibility that the identified AUF1 interacting proteins might be involved in the regulation of mRNA stability mediated by AUF1.

Y-Box Binding Protein 1 and RNase UK114 Mediate Monocyte Chemoattractant Protein 1 mRNA Stability in Vascular Smooth Muscle Cells

“…Although our data indicate that GR and YB-1 both play an important role in mediating MCP-1 mRNA degradation, neither molecule is "classically" a nuclease. There is a single report that YB-1 has endoribonuclease activity (against a synthetic 38-base oligomer) in vitro (34). Although SMC extracts could degrade the 38-mer, we were not able to block degradation with the YB-1 Ab under conditions in which it blocked MCP-1 mRNA degradation by the same extracts (data not shown).…”

“…Of particular note were YB-1 and UK, both of which have been implicated in regulating mRNA expression and/or stability. YB-1 is a DNA/RNA-binding protein shown to regulate transcription and translation (25) and has also been reported to possess endoribonuclease activity (34). UK is an endoribonuclease that has been shown to inhibit translation by cleaving mRNA (33).…”

Proteomic Characterization of Messenger Ribonucleoprotein Complexes Bound to Nontranslated or Translated Poly(A) mRNAs in the Rat Cerebral Cortex

“…Of special interest is also the repeated detection of protein similar to ubiquitin C-terminal hydrolase within mRNP complexes, which is likely involved in the processes of de-ubiquitination. Whether this protein is part of a specific signaling pathway or an element controlling the ubiquitination and function of ribosomal proteins, such as L40, S27a P1, translation factors, or AUF1 (22)(23)(24)(25), remains to be investigated. The presence of two enzymes involved in glycolysis (phosphofructokinase C and glyceraldehyde-3-phosphate dehydrogenase) in an mRNP preparation was not expected but may support the assumption that there exists a link between glycolysis and the protein synthesis machinery (26).…”

The role of AUF1 in thyroid carcinoma progression

“…Some differences in the activity of various AUF1 isoforms are reported, but they generally enhance the degradation of target mRNAs in connection with ubiquitin and/or a proteasome complex , Laroia et al 2002, Moraes et al 2003.…”