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.