You'd Prefer An Argonaute

An RNA-dependent RNA polymerase formed by TERT and the RMRP RNA

Yoshiko Maida, Mami Yasukawa, Miho Furuuchi, Timo Lassmann, Richard Possemato, Naoko Okamoto, Vivi Kasim, Yoshihide Hayashizaki, William C. Hahn & Kenkichi Masutomi

Nature AOP, 23 August 2009.
doi:10.1038/nature08283

This week’s summary and sound analysis by Jenny Rood:

Summary:  The Masutomi lab, in collaboration with others, present evidence in this article for a novel functionality of the human telomerase reverse transcriptase catalytic subunit, hTERT, when it is bound to the RNA RMRP.  TERT has previously been shown to form a complex with the TERC RNA, providing the template for telomere elongation. This paper demonstrates the interaction of TERT and RMRP in vitro and in vivo and then argues that this complex serves as a RNA-dependent RNA polymerase whose eventual function is to synthesize double-stranded RMRP RNA for the siRNA pathway.

The authors first identify the TERT-RMRP interaction through immunoprecipitation of overexpressed tagged hTERT and then isolation and sequencing of the associated RNAs.  RMRP sequences appeared roughly as frequently as TERC sequences, together making up 5% of hits; a total of 38 RNA sequences were found to be associated with TERT in this assay.  This interaction is then confirmed by RT-PCR and northern blot.  Purifications with a variety of overexpressed TERT fragments show that the RNA interaction occurs in the N-terminal half of the protein, where TERC is also known to be bound, yet the TERT-RMRP complex fails to elongate telomeres in the PCR-based TRAP assay.

Under high salt conditions (approximately double physiological salt) in vitro, the authors are able to isolate an RNA species that is twice the length of the RMRP RNA that reacts with both sense and antisense probes to RMRP.  Moreover, truncation of the 3’ end of RMRP eliminates this product, supporting a 3’ back-priming mechanism and implicating TERT-RMRP as a RNA-dependent RNA polymerase (RdRP).  Cell lines lacking TERT (VA-13) also fail to form the double-stranded product, but overexpression of TERT in these lines rescues this phenotype.

Overexpression of RMRP, on the other hand, in cell lines containing TERT leads to a reduction in RMRP signal.  Short RNAs complementary to RMRP can also be isolated from these cell lines, implicating an siRNA mechanism.  Further evidence for the generation of RMRP siRNA through the RdRP activity of TERT-RMRP is provided by association of the short RMRP species with Argonaute 2 and rescue of the RMRP overexpression phenotype in Dicer knockdowns.

Comments and future directions:  The paper clearly demonstrates an interaction between TERT and RMRP RNA, and progresses towards a possible model for the function of this RdRP, but many questions remain.  The experiments showing the generation of siRNAs and their effects in vivo seem preliminary and rushed.  During discussion of this paper in journal club, it was mentioned that figure 4e might have been improved by including a negative control: does the VA-13 cell line, which lacks TERT, also lack RMRP small RNAs?

The paper mentions that both TERC and RMRP bind in the same region, but this region is defined very broadly as the N-terminal half of TERT.  It would be very interesting to have more structural evidence about the interaction of TERT with these two RNAs.  Are the same residues responsible for the binding of both RNAs?  Are these two RNA binding events mutually exclusive?  If so, what implications does this have for the function of TERT?

A known set of mutations in RMRP is responsible for the human disorder of cartilage-hair hypoplasia.  It would be interesting to determine if these mutations inactivate the TERT-RMRP RdRP activity, and how in turn this causes a pleiotropic disease.

Finally, on a broader note, the authors discuss that a back-priming mechanism inherently limits the number of possible products (verified by incubating TERT-RMRP with total RNA).  It would be useful to determine which products are made, and consequently, if these are also processed into siRNA, or what function they serve.  Similarly, it would be interesting to see if any other RNAs from the initial TERT screen for bound RNAs had similar functions to the TERT-RMRP complex.

In summary, this paper suggests a tantalizing new function for TERT in complex with a different RNA besides the canonical TERC that will likely provide many insights in the future.

Co-translational mRNA decay in Saccharomyces cerevisiae

Wenqian Hu, Thomas J. Sweet, Sangpen Chamnongpol, Kristian E. Baker & Jeff Coller

Nature 461 (7261): 225-229, 10 September 2009.
Nature AOP, 23 August 2009.
doi:10.1038/nature08265

This week’s exacting summary and analysis by David Weinberg:

In their recent Nature article, Jeff Coller and colleagues demonstrate that mRNA in decay in the budding yeast Saccharomyces cerevisiae can occur while the mRNA is still engaged with actively translating ribosomes.  Prior to this paper, the dogma in the field had been that ribosome dissociation was a necessary step before decapping.  The evidence for this exact model was a bit lacking: it was clear that translation initiation and decapping are competing processes since both require access to the cap structure, but there didn’t seem to be any indication that decapping would have to interfere with elongating ribosomes.  Indeed, co-translational mRNA decay had been previously hypothesized but no lab had demonstrated it.  Using the tools of budding yeast and some clever molecular biology, Jeff Coller’s lab is able to do just that.

The basic outline of the article is a series of very similar experiments showing that decaying mRNAs (i.e., deadenylated, decapped, and/or partially degraded) are associated with translating ribosomes using different combinations of knock-out and wild-type strains, and artificial and endogenous mRNAs.  The initial indication that the existing model for mRNA decay might be incorrect is that deadenylated mRNAs that accumulate in a decapping-defective strain remain on polyribosomes.  The same is true of decapped mRNAs that accumulate in an XRN1 knock-out strain.  The authors are careful to include enough control experiments to show that the polyribosome association suggested by sucrose gradients is actually due to bound ribosomes and not another macromolecular complex that might similarly alter the sedimentation properties of an mRNA.

Next, using both transcriptional turn-on and shut-off experiments the authors claim to show that decapping can occur when mRNAs are associated with translating ribosomes.  This point – that the associated ribosomes are actively translating when decapping occurs – is important to distinguish from ribosome-reloading following mRNA decapping.  The turn-on experiment is a bit bogus as their interpretation is based on the observation that at the first timepoint when a decapped mRNA accumulates, that decapped mRNA is on polyribosomes.  Given that they had 2 timepoints (20 and 60min), this conclusion seems like a stretch and could have been left out of the paper without any detriment.  However, the transcriptional shut-off experiment is convincing: adding cycloheximide at the same time that transcription is shut-off prevents the ribosome run-off on decapped mRNAs that is seen in the absence of cycloheximide.  Thus, the ribosomes observed to be associated with decapped mRNAs were in the act of translation.

Up to this point, all experiments had been performed in mRNA decay mutants in order to observe relatively rare decay intermediates.  Using an artificial mRNA in which rare codons are used to stall translating ribosomes, the authors demonstrate that their conclusions also hold in wild-type cells.  Of course, such an artificial mRNA can potentially give results that aren’t true of endogenous mRNAs that are efficiently translated.  The authors therefore conclude their paper with the ultimate demonstration of co-translational mRNA decay for two different (highly expressed) endogenous mRNAs in wild-type cells.

From their work, the authors conclude that a ribosome-free state is not required for mRNA decay and, in fact, mRNA decay can occur co-translationally.  This ability to initiate decay of mRNAs that are still being translated seems to provide a more rapid means of mRNA decay: rather than waiting for translation to finish or ribosomes to be actively removed, Xrn1 can begin to degrade the mRNA from the 5′ end immediately after decapping.  The paper also makes an evolutionary argument that such a decay mechanism would have the benefit that it would not interfere with residual translating ribosomes and, therefore, would prevent the production of truncated polypeptides.

While the science in this paper is (for the most part) convincing, its presentation is a bit frustrating.  At the end of the paper, the experiments that the reader should really care about – those performed with endogenous mRNAs in wild-type cells – are FINALLY shown.  But to get there the reader has to get through the artifact-prone experiments done in other settings.  Of course, these artifact-prone experiments lend further support to the final model.  However, I would have rather seen some space in the paper devoted to further characterization of the decay mechanism.  For example:
– Do the same principles hold for all mRNAs?  In particular, is this true of histone mRNAs that lack poly-A tails? Lower expressed mRNAs that are more difficult to detect?
–  Is Xrn1 decay of ribosome-associated mRNAs distributive or processive?  Are there cycles of degradation/dissociation as ribosomes finish elongating, or does Xrn1 remained engaged throughout?
– How does mRNA half-life relate to ribosome occupancy?  Is slow translation of an mRNA associated with slow decay?
I hope that we can find some answers to these questions in future work from the Coller lab.

Architecture and secondary structure of an entire HIV-1 RNA genome

Joseph M. Watts, Kristen K. Dang, Robert J. Gorelick, Christopher W. Leonard, Julian W. Bess Jr, Ronald Swanstrom, Christina L. Burch & Kevin M. Weeks

Nature 460 (7256): 711-716, August 2009.
doi:10.1038/nature08237

This week’s snappy summary and analysis by Vikram Agarwal:

In this article, Watts and colleagues demonstrate a method to systematically predict the RNA secondary structure of an HIV-1 viral genome.  Their technique, called SHAPE (2′-hydroxyl acylation analysed by primer extension), exploits the principle that nucleotides in a flexible conformation can react with an electrophile that subsequently blocks a primer extension reaction.  In this way, one can visualize and quantify the tendency of regions of RNA to participate in base pairing or remain unstructured, all at single-nucleotide resolution.  The technique was recently shown to accurately predict nearly 95-100% of bases in rRNA, tRNA, and several coding RNAs (Deigan et al., PNAS 2009).  The SHAPE reactivity of each base position is converted linearly into a pseudo-free energy term, which is incorporated into RNAstructure to fold the entire RNA.

Here the foldings reconstruct known motifs in the HIV genome, such as the gag-pol frameshift element, which are shown here to actually be constituents of more extensive motifs.  Most notably, the technique allows the group to predict an accurate structure for all coding regions, which have been formidable to characterize using traditional approaches.  The paper finds significant correlations between RNA secondary structure and protein secondary structure, with inter-protein linkers and protein-domain junctions often corresponding to highly structured RNA regions.  This result implicates such highly structured regions in modulating and offering time for protein folding during translation.  To investigate this possibility, the authors perform a ribosomal toeprinting experiment on two HIV-1 open-reading frames to test the hypothesis that local RNA flexibility influences the pausing of a ribosome as it scans the RNA message (Supplementary Figure 5).

Overall, this paper contributes a notable advance in the accurate characterization of RNA structures on a large scale.  It opens the door for parallelization of SHAPE analysis to characterize even larger RNA genomes at high-resolution, which would open a wealth of knowledge about how RNA motifs and other signals are interpreted by the cell.  Moreover, it suggests a link between RNA structure and protein folding.  However, this link requires a more thorough and direct investigation in the future.  The authors establish only a correlative relationship between the RNA and protein structure, and have yet to dissect the underlying causality of the process.  This may ultimately merit mutational experiments that modify RNA secondary structure to examine if regions within a protein are differentially folded via the fine-tuning of ribosomal processivity by the base pairing interactions of RNA.