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Mitotic cell-cycle progression is regulated by CPEB1 and CPEB4-dependent translational control

Isabel Novoa, Javier Gallego, Pedro G. Ferreira  &  Raul Mendez

Nature Cell Biology 12: 447 – 456, May 2010.
doi:10.1038/ncb2046

This week’s lucid summary and analysis by Noah Spies. This is Noah’s second contribution to the blog:

A normal eukaryotic messenger RNA is capped, spliced and polyadenylated within the nucleus in preparation for export into the wild world of the cytoplasm. Once in the cytoplasm, the translational machinery will recognize the cap and poly(A) tail and recruit ribosomes to translate the message until its eventual decapping/deadenylation and decay. In the late 1980s, it was discovered that many messages in Xenopus oocytes lay dormant in the cytoplasm until oocyte maturation or fertilization. These messages are recognized by a Cytoplasmic Polyadenylation Element (CPE) in their 3′ UTR by the CPE binding protein CPEB, and are deadenylated in the cytoplasm and stored in a translationally inert form until activation and re-adenylation at some later time point. Previous in vitro experiments suggested this system may activate some messages during the embryonic cell cycle, but there was little follow-up on these old results prior to this paper.

Novoa, et al (2010) used a differential elution system to purify mRNAs with short poly(A) tails (<30bp) and compared this pool to total mRNA (poly(A) tail length > 9bp) using microarrays. The authors isolated HeLa cells in S phase and compared these to cells in G2/M phases. The microarray experiment, largely validated by PCR (75% concordant results), showed several hundred genes whose poly(A) tails were either longer or shorter in G2/M than in S phase, and polysome profiling confirmed translational changes for a handful of these.

To connect these results to cytoplasmic polyadenylation, Novoa and colleagues showed the CPEB1 and CPEB4 were expressed in HeLa cells and CPEB1/2/4 were functional in an in vitro binding assay. Upon shRNA knock-down of CPEB1 and CPEB4, a number of genes showed differential polyadenylation profiles by microarray, hinting at a possible global role for CPEBs in regulating genes during the cell cycle. Experimental follow-up on one of these hits showed shRNA knock-down of CPEB lengthened the poly(A) tail of the Mnt mRNA, and this lengthening increased the protein levels. Other genes (e.g. CDKN3) showed similar effects.

If there are so many genes regulated by CPEB1/4 during the cell cycle, it should follow that there would be a significant phenotype following knock-down of these factors. Indeed, the authors found cell proliferation defects in both knock-downs, but not in a control knock-down. Much FACS analysis later, the authors conclude that CPEB knock-down results in a mitotic entry defect.

This work suggests that there is yet another layer of gene regulation operating during the cell cycle, and like any good research, raises more questions than it answers.

First, poly(A) tail lengths clearly change after CPEB knock-downs, but it remains an open question how many of these effects are direct. The authors attempted a cursory motif enrichment analysis, but by their own admission, this was limited in its scope by the loose definition of these elements. Differentially polyadenylated genes were enriched for a number of regulatory motifs (CPE, ARE, microRNA), suggesting that some of the changes might have been due to effects other than cytoplasmic polyadenyation. A simple follow-up would be to express a reporter containing a putative regulated 3′ UTR and follow poly(A) tail length after CPEB knock-down or after mutation of the CPE element. Further work might include immunoprecipitation of CPEB-bound RNAs at different cell cycle stages to confirm a direct interaction. And the motif work could be expanded upon with more specific control sets (e.g. only expressed genes) and with only microRNAs expressed in HeLa cells (it’s unclear how this was done).

Secondly, it is important to identify which CPEB interactions are important for the cell proliferation defect, as it is plausible that such an effect could result from shRNA off-target effects.

Finally, the CPEB1 knock-out mouse is viable, though sterile. If CPEB1 plays an important role during the cell cycle, this suggests that CPEB4 may be partially redundant with CPEB1. Seemingly inconsistent with this result, CPEB1 and CPEB4 knock-downs show only a small proportion of the same regulated genes.

Citation for Research Blogging:

Novoa I, Gallego J, Ferreira PG, & Mendez R (2010). Mitotic cell-cycle progression is regulated by CPEB1 and CPEB4-dependent translational control. Nature cell biology, 12 (5), 447-56 PMID: 20364142