PUF3 Acceleration of Deadenylation in Vivo Can Operate Independently of CCR4 Activity, Possibly Involving Effects on the PAB1–mRNP Structure

Abstract

The evolutionarily conserved PUF proteins stimulate CCR4 mRNA deadenylation through binding to 3′ UTR sequences of specific mRNA. We have investigated the mechanisms by which PUF3 in Saccharomyces cerevisiae accelerates deadenylation of the COX17 mRNA. PUF3 was shown to affect PAN2 deadenylation of the COX17 mRNA independent of the presence of CCR4, suggesting that PUF3 acts though a general mechanism to affect deadenylation. Similarly, eIF4E, the cap-binding translation initiation factor, known to control CCR4 deadenylation, was shown to affect PAN2 activity in vivo. PUF3 was found to be required for eIF4E effects on COX17 deadenylation. Both eIF4E and PUF3 effects on deadenylation were shown, in turn, to necessitate a functional poly(A) binding protein (PAB1) in which removal of the RRM1 domain of PAB1 blocked both their effects on deadenylation. While removal of the proline-rich region (P domain) of PAB1 substantially reduces CCR4 deadenylation at non-PUF3 controlled mRNA and correspondingly blocked eIF4E effects on deadenylation, PUF3 essentially bypassed this P domain requirement. These results indicate that the PAB1-mRNP structure is critical for PUF3 action. We also found that multiple components of the CCR4-NOT deadenylase complex but not PAN2 interacted with PUF3. PUF3 appears, therefore, both to act independently of CCR4 activity, possibly through effects on PAB1-mRNP structure, and to be capable of retaining the CCR4-NOT complex.

Quantitative proteomic analysis reveals concurrent RNA–protein interactions and identifies new RNA-binding proteins in Saccharomyces cerevisiae

“…DNA-binding and transcription-related GO terms were also enriched among proteins that we found to have RNAdependent interactions with Nab2 or Pab1, but not with Puf3-further evidence that Pab1 and Nab2 bind cotranscriptionally to nascent transcripts, but that Puf3 does not. Conversely, the KEGG RNA degradation pathway annotation was specifically enriched among the proteins interacting in an RNA-dependent manner with Puf3, consistent with the known role of Puf3 in promoting the degradation of its mRNA targets (Gerber et al 2004;Lee et al 2010). …”

“…Puf3 promotes the decay and localization of its mRNA targets (Gerber et al 2004;Saint-Georges et al 2008;Lee et al 2010;Quenault et al 2011). It also physically interacts with decay proteins such as the major cytoplasmic deadenylase complex Ccr4-Not (in an RNAindependent manner) (Lee et al 2010).…”

Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site

“…These proteins bind elements in 3′ untranslated regions (3′UTRs), termed PUF binding elements (PBEs) (1,2). PUFs commonly repress translation or enhance mRNA decay (3)(4)(5)(6)(7)(8) but also can activate and localize mRNAs (7,(9)(10)(11)(12)(13)(14). The six PUF proteins of Saccharomyces cerevisiae control distinct sets of RNAs that comprise distinct functional groups and bind ∼850 mRNAs, or 10-15% of the mRNA species in that organism (15,16).…”

The Pumilio-domain protein PUF6 contributes to SIDER2 retroposon-mediated mRNA decay in Leishmania

“…In humans, PUF proteins, PUM1 and PUM2, recruit the CCR4-NOT complex to stimulate mRNA decay and repress translation (Van Etten et al 2012). In S. cerevisiae, recruitment of CCR4p, the catalytic subunit of the CCR4-POP2-NOT deadenylase complex, by PUF3p, PUF4p or PUF5p, results in mRNA deadenylation (Goldstrohm et al 2006Hook et al 2007;Lee et al 2010). A similar mechanism has been reported in Drosophila, C. elegans, and humans, where recruitment of the deadenylase complex POP2p subunit by PUF proteins stimulates deadenylation of target mRNAs (Goldstrohm et al 2006;Kadyrova et al 2007;Suh et al 2009).…”