Studying the role of TERRA in telomere protection and length regulation

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PROJECT SUMMARY Telomeres are specialized DNA-protein complex that protect the ends of linear chromosomes. Mammalian telomeres are composed of highly conserved tandem repeat sequences of duplexed (TTAGGG)n, ending with a 3’ single stranded (TTAGGG)n sequence that is bound to telomere specific proteins known as the shelterin complex. In healthy somatic cells, progressive loss of telomeres results in cellular senescence. In 85% of cancer cells, however telomeres are elongated by telomerase, while the remaining 15% of cancers use alternative lengthening of telomeres (ALT). ALT pathway leads to overexpression of Telomeric repeat containing RNA (TERRA), a long noncoding RNA that is transcribed from telomeres. TERRA is capable of trans annealing by invading the telomeric duplex to form an R-loop (DNA-RNA hybrid) structure. Consistently, the accumulation of R-loops is one of the hallmarks of ALT cancer. In addition, a recent study showed that R-loop formation is promoted by Rad51 recombinase, but it is antagonized by RNase H1. Despite the mounting evidence of TERRA’s critical role in telomere structure and function, our molecular understanding of TERRA is limited. Here, I propose to elucidate the molecular mechanism underlying TERRA recruitment and invasion into telomeres in the context of shelterin proteins and the extent to which TERRA regulates telomere length in cells. Aim 1 will probe TERRA-induced R-loop formation and the role of shelterin proteins. My preliminary results using single molecule (sm) FRET show that TERRA trans anneals to telomeric DNA, and unexpectedly, the resulting R-loop exhibits dynamic movement that is stabilized by TRF2. I also employed an sm-colocalization assay to measure the efficiency for trans annealing of TERRA to telomeric DNA. My results from this assay show that the presence of non-TERRA sequence, which represents the subtelomeric DNA, as well as the presence of G-quadruplex (G4) telomeric overhang significantly enhances TERRA binding to telomeric duplex. Aim 2 will elucidate the mechanism underlying TERRA recruitment and invasion by Rad51 and shelterin proteins. I present my preliminary results using an sm-colocalization assay which show that RAD51-TERRA complex invades telomere duplex more efficiently than TERRA alone. Aim 3 will measure telomere length as a function of TERRA levels in ALT- and telomerase dependent cancer cells. Additionally, this aim will study the role of TRF2, POT1, Rad51, RNaseH1 in TERRA regulation of telomeres in cells. Completion of these aims will provide a molecular mechanism underpinning TERRA function in telomere protection and length regulation and thereby help develop treatment for ALT positive cancers in which TERRA is upregulated.