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Final Report Summary - HTR (Towards the structural understanding of human telomerase)

It has been clear for some years that both DNA and RNA play important roles in biology other than storing the genetic information and allowing it to be transcribed to proteins, the molecular machines. In a way, DNA does not only store information in its nucleotide sequence or through epigenetic factors such as methylation, but also in its structure. Indeed the diversity of structures DNA can form extends far beyond canonical duplex DNA and is reflected in their influence in many cellular processes.

However, RNA has even greater potential to form stable and complex three dimensional structures providing rich opportunities to perform complex chemistry and biology in the cell. The combination of both protein and RNA structures can provide additional opportunities for function. Our focus is on the ribonucleoprotein telomerase, a specialised enzyme that can elongate the ends of linear chromosomes by adding tandem repeats of guanine rich sequences to the 3’ end of telomeres.

Telomeres are repetitive nucleotide sequences rich in guanine residues located at the termini of linear chromosomes of most eukaryotic organisms. Telomeres function as capping agents to protect chromosomes both from shortening at each replication cycle and from fusing with other chromosome ends or rearranging. Maintenance of telomeres length is critical for genomic stability and cell viability. Telomeres are consumed during cell division but can be replenished by telomerase, a unique ribonucleoprotein enzyme that adds telomeric repeats onto chromosome ends to maintain telomere length. Telomerase uses an RNA template that is integral part of the enzyme and a specialized reverse transcriptase to processively synthesize the G-rich telomeric strand. The importance of the discovery of telomeres and telomerase was recognized by the Nobel Prize award in Physiology or Medicine in 2009.

Telomerase is not active in normal somatic cells, but is highly active in most cancers, and is thus of interest as a target for anticancer drugs. In addition, mutations in both the telomerase RNA and telomerase proteins are associated with some diseases. Human telomerase contains a 451 nt RNA along with a variety of proteins besides the reverse transcriptase. Only a few nucleotides of human telomerase RNA (hTR) are needed as a template to elongate telomeres, but the function of all the rest of the RNA is mostly unknown.

Despite the importance of human telomerase in stem cells, cell death, aging and cancer, no accurate structure exists. This project focused on the RNA part of human telomerase (human telomerase RNA or hTR). The objectives were to determine by X-ray crystallography the 3D structure of separate domains of hTR, the interaction between domains and, ultimately, the structure of the whole hTR. The knowledge of the precise three-dimensional structure of the hTR and its interaction with telomerase reverse transcriptase is essential to understand its role in telomerase function and how it can be regulated.

The objective to generate diffraction quality crystals was very ambitious and, unfortunately, could not be accomplished during the two-year duration of the project. As planned, we prepared several RNA constructs using the previous expertise of the fellow and the vast knowledge of the scientist in charge about crystallisation of nucleic acids. RNA constructs included modifications of the domain sizes and ends, introduction of the GAAA tetraloop as a crystallisation module to the full-length RNA, and introduction of the U1A binding site to set up crystallisations with the spliceosomal protein U1A double mutant (Y31H, Q36R). These last crystallisations with the U1A protein-RNA complex were delayed due to technical issues in the last step of purification of the protein. Crystallisation trials for the other constructs were set both using robotic facilities for screening and by-hand to produce diffraction-quality RNA crystals, but our efforts were unsuccessful. However, crystallisation trials are still running and further investigation will continue in the laboratory of Dr Gary N Parkinson.

In order to achieve the training objectives for the fellow, we also investigated the detailed atomic structure of the complementary sequence of human telomere repeats (TTAGGG) that can fold into i-motif structures. We chose three different sequences with a different number of repeats (one, two and four repeats) that had been previously crystallised but unpublished, and as a learning tool we attempted to solve them using standard molecular replacement techniques. However, the available structural models proved inadequate requiring direct phasing using brominated analogues allowing the collection of anomalous data on the Br. This would allow us to obtain the position of the bromine atom and from here derive phases and solve the phase problem. Although several modified sequences were assessed, this method initially proved unsuccessful as the substitutions require new crystallisations and further data collections. New crystals and several dataset containing anomalous data were collected and a partial model was determined. Finally, we were able to solve the structure for the one-repeat i-motif by acquiring additional anomalous data on the native phosphorous atoms of the original sequence on the newly created long-wavelength beamline at DLS (I23). The scientists at DLS combined the phase information from the Br derivative and P atoms and then used their strong anomalous diffraction for the phasing resulting in the determination a high-resolution i-motif structure containing the human telomeric repeat. This structure is now available to solve by molecular replacement the structures of the two and four-repeats. This work is close to completion and will be published in due time.

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Malgorzata Kielbasa, (European Contracts Manager)
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