Structure of Long Telomeric RNA Transcripts

Abstract

Recent studies have identified RNA transcripts arising from mammalian telomeres with the transcript of the C-rich strand (r(5-UUAGGG-3) n ) being much more abundant than transcripts of the G-rich strand. Here we used transmission electron microscopy, CD, and nuclease digestion to investigate the structure of ϳ640-nucleotide (nt) RNA transcripts of the C-rich and G-rich strands of mammalian telomeric DNA. The CD spectrum of the C-rich RNA in low salt (10 mM KCl) or high salt (100 mM KCl) was typical of mixed sequence RNA, whereas the CD spectrum for the G-rich RNA differed with changes characteristic of parallel G-quadruplexes at the higher salt concentration. Electron microscopy visualization of the C-rich RNA revealed relatively extended unstructured molecules 59.7 ؎ 17.8 nm in length and with a width consistent with single-stranded RNA following metal coating. In contrast, the G-rich RNA was observed as round particles and short, thick rods with the rods being most prevalent in high salt conditions and absent in low salt. The rods were 22.7 ؎ 4.8 nm in length and 7.6 nm in width. Digestion of the G-rich RNA with T1 RNA nuclease revealed a ladder of bands whose sizes were integral multiples of 24 nt plus a 4-nt overhang. These observations suggest a model in which G-rich telomeric RNA folds into chains of particles each consisting of four (UUAGGG) repeats stabilized by parallel G-quartets and joined by UUA linkers. These chains further condense to form short rods and round particles.Telomeres are the terminal elements of eukaryotic chromosomes. They maintain the integrity of linear chromosomes by blocking misrecognition of the DNA ends as double strand breaks. They also provide a mechanism to overcome the progressive erosion of DNA sequences at chromosome ends due to the end replication problem, and they serve to control transcription of genes near the telomere. These complex functions reflect the interplay between a large set of both telomere-specific and nonspecific factors and the unique structural organization of the telomere that sets it apart from the remainder of the genome. Indeed telomeres have long been known to be heterochromatic and to show telomere position effects in which transcription of genes located near the telomere is repressed (reviewed in Ref. 1).Telomeric DNA of most higher members of the animal kingdom consists of ϳ5-20 kb of the repeat (5Ј-TTAGGG-3Ј) n , whereas in plants, the repeat is (5Ј-TTTAGGG-3Ј) n with plant telomeres frequently exceeding 50 kb (2, 3). The normal cellular core histones comprise the major protein species at plant and animal telomeres. In addition, there are six telomere-specific proteins termed the shelterin proteins that have been identified largely in mammalian cells, and they consist of TRF1 and TRF2, which bind directly to duplex telomeric repeats, Pot1, which binds to the single-stranded 3Ј overhang on the G-rich strand, and additional factors including hRap1, Tin2, and TPP1, which scaffold onto TRF1 and TRF2 (reviewed in Ref. 4).The organization of telomeres...

Mechanical unfolding of long human telomeric RNA (TERRA)

“…This result is in agreement with previously reported observations showing that other telomeric RNA sequences are folded into parallel G-quadruplex structures both in sodium and potassium solutions. 12,13,[16][17][18][19][20] It is also important to mention that no band at 300 nm was observed for this long telomeric RNA as it was reported for 22-mer and 45-mer telomeric RNA in ammonium acetate buffer. 15 On the other hand, the normalized CD spectra of the bimolecular G-quadruplex formed by r(UAGGGUUAGGGU) and Q4 showed a similar shape and amplitude, which indicates that most of the Q4 molecules form four G-quadruplexes (Fig.…”

The 5′ Guanosine Tracts of Human Telomerase RNA Are Recognized by the G-Quadruplex Binding Domain of the RNA Helicase DHX36 and Function To Increase RNA Accumulation

“…Synthetic RNAs harboring on May 9, 2018 by guest http://mcb.asm.org/ hTR sequences 5Ј of the template can fold as a G-quadruplex (19,26). Importantly, unlike DNA, RNA uniformly adopts the parallel-strand orientation of quadruplex in vitro and in vivo (4,8,30,41). DHX36 has the ability to bind and resolve a model intermolecular G-quadruplex RNA (11).…”

“…Helicase domain proteins participate broadly in RNA processing, RNP biogenesis, and RNP regulation with strand-separating, strand-annealing, and RNP-remodeling activities potentially relevant to telomerase RNP maturation and function (24). DHX36 in particular has several potentially relevant substrates, because model oligonucleotides with the sequence of human telomeric DNA, telomeric RNA, or the 5Ј region of hTR can form Hoogsteen-base-paired G-quadruplex structures in vitro (4,8,19,26,30,41). We therefore investigated whether DHX36 associates with human telomerase and, if so, whether the interaction has defined sequence requirements or a more chaperone-like general specificity.…”

Unraveling secrets of telomeres: One molecule at a time

“…This result suggests that G-quadruplex structure may form in vivo at blunt telomeric ends, providing an alternative protective structure at telomeres. In addition, G-rich telomeric RNA was observed as round particles or short, thick rods in EM micrographs supporting the idea that G-rich RNA in TERRA folds into a string of G-quadruplex beads [67]. Telomeric RNA can also directly interact with telomeric DNA to form hybrid intermolecular G-quadruplexes, adding another layer of complexity to the telomere regulation [68].…”