CUG-BP and 3'UTR sequences influence PARN-mediated deadenylation in mammalian cell extracts

Abstract

Several mRNAs have been shown to exhibit distinct patterns of poly(A) shortening prior to their decay in vivo. In this study, we show that individual transcripts also demonstrate distinct patterns of deadenylation in in vitro systems derived from HeLa and Jurkat T cell cytoplasmic extracts. The major patterns observed were slow/synchronous and fast/asynchronous poly(A) tail shortening. For all RNA substrates tested, PARN was shown to be the enzyme responsible for the deadenylation patterns that were observed. Sequences in the 3' untranslated regions influenced the deadenylation pattern. Using a fragment of the 3'UTR of the c-fos mRNA as a model, the interaction of CUG-BP, the human homolog of EDEN-BP -a protein previously implicated in regulated deadenylation in Xenopus oocyteswas shown to be associated with changes in PARN-mediated deadenylation patterns. Our results suggest that association of CUG-BP with 3'UTR sequences can modulate the activity of the PARN deadenylase in mammalian cell extracts.

Structural insights into the targeting of mRNA GU-rich elements by the three RRMs of CELF1

“…Structures derived from Raver1 peptide-RRM complexes reveal the versatility of RRMs as a recognition motif in mediating protein interactions across the distal helical face while engaged in RNA binding across the β-sheet. By analogy, recent studies using in vitro assays for assessing mRNA decay in AU-rich mRNAs have shown that CELF1 binds specifically to mRNAs and stimulates poly(A) shortening in the 3′ UTR by recruiting the poly(A) deadenylase PARN (13,30). Although the details of this interaction are still ill-defined, it would appear that a Ser-rich motif within the RRM2-RRM3 flexible linker region of CELF1, close to a putative amphipathic helical motif (residues 332–340), facilitates recruitment.…”

“…In Xenopus , CELF1 induces deadenylation and appears to play a key role in regulating poly(A) shortening (27–29). Recent studies, using in vitro assays for assessing mRNA decay in AU-rich mRNAs, have shown that CELF1 binds specifically to mRNAs and stimulates poly(A) shortening in the 3′ untranslated region (3′ UTR) by recruiting the poly(A) deadenylase ribonuclease (PARN) (13,30). In this case, binding to target EDEN mRNA GU-rich elements (GREs) is necessary for function (2,12,31–35), with EDEN-dependent deadenylation regulated by neighbouring cis -acting AU-rich elements (AREs) (13).…”

“…The same study reported in vitro assays which show that CELF1 enhances deadenylation of these RNA substrates. The observation that the poly(A) deadenylase PARN is co-immunodepleted with CELF1 strongly suggests that PARN and CELF1 form a protein-mediated complex (30). Glutathione S-transferase (GST) pull-down assays and detection by western blotting confirmed that the two proteins have a direct co-interaction, and this may be regulating deadenylation rates, rather than binding to a common RNA substrate, increasing processivity and poly(A) shortening.…”