4492–4506 Nucleic Acids Research, 2020, Vol. 48, No. 8 Published online 4 March 2020 doi: 10.1093/nar/gkaa134 Structure specific recognition of telomeric repeats containing RNA by the RGG-box of hnRNPA1 Meenakshi Ghosh 1 and Mahavir Singh 1,2,*
1Molecular Biophysics Unit, Indian Institute of Science, Bengaluru, 560012, India and 2NMR Research Centre, Indian Institute of Science, Bengaluru, 560012, India
Received April 12, 2019; Revised February 12, 2020; Editorial Decision February 19, 2020; Accepted February 21, 2020 Downloaded from https://academic.oup.com/nar/article/48/8/4492/5780085 by guest on 25 September 2021
ABSTRACT mation, and replication (4,5). TERRA RNA repeats have been found to bind a number of proteins that include telom- The telomere repeats containing RNA (TERRA) is erase reverse transcriptase (hTERT), telomeric repeat bind- transcribed from the C-rich strand of telomere DNA ing factor-2 (TRF2) and heterogenous nuclear ribonucleo- and comprises of UUAGGG nucleotides repeats in protein A1 (hnRNPA1) (6,7). Complexity of TERRA func- humans. The TERRA RNA repeats can exist in single tions also stems from the observation that TERRA re- stranded, RNA-DNA hybrid and G-quadruplex forms peats can exist in single-stranded, RNA-DNA hybrid, and in the cell. Interaction of TERRA RNA with hnRNPA1 RNA G-quadruplex forms in the cell (8). The G-quadruplex has been proposed to play critical roles in mainte- forms can further exist in structures of different topologies nance of telomere DNA. hnRNPA1 contains an N- with intramolecular G-quadruplex being the most physio- terminal UP1 domain followed by an RGG-box con- logically relevant form. Therefore, how a TERRA binding taining C-terminal region. RGG-motifs are emerging protein recognizes the TERRA sequences in different con- formations is an important question. as key protein motifs that recognize the higher or- hnRNPA1 is an abundant protein in the eukaryotic cell der nucleic acid structures as well as are known to that is involved in telomere DNA maintenance apart from promote liquid-liquid phase separation of proteins. its roles in RNA splicing, stability and transport (9–11). The In this study, we have shown that the RGG-box of N terminus of hnRNPA1 consists of two RNA binding do- hnRNPA1 specifically recognizes the TERRA RNA mains, namely RRM1 and RRM2, collectively called the G-quadruplexes that have loops in their topology, UP1 domain. UP1 is followed by a C-terminal region that whereas it does not interact with the single-stranded contains an RGG-box, a prion-like domain, and a M9 nu- RNA. Our results show that the N-terminal UP1 do- clear shuttling sequence (Figure 1A). In RGG-box contain- main in the presence of the RGG-box destabilizes ing proteins, typically two RGG repeats are separated by 0– the loop containing TERRA RNA G-quadruplex effi- 4 intervening amino acid residues, though there are also re- ciently compared to the RNA G-quadruplex that lacks ports of RGG repeats being separated by up to nine residues (12–14). In hnRNPA1, the RGG-box consists of four RGG loops, suggesting that unfolding of G-quadruplex repeats comprising of a tri-RGG motif (RGG-X4-RGG-X4- structures by UP1 is structure dependent. Further- RGG, where X is any amino acid) and a single RGG repeat more, we have compared the telomere DNA and that is present nine amino acids away towards the N termi- TERRA RNA G-quadruplex binding by the RGG-box nal region of the tri-RGG motif (Figure 1B). of hnRNPA1 and discussed its implications in telom- The UP1 domain has been shown to interact with both ere DNA maintenance. telomere DNA and TERRA RNA repeats and regulate the telomerase activity. These interactions have been proposed to have multiple roles pertaining to telomere DNA pro- INTRODUCTION tection (15–21). Since TERRA RNA repeats are comple- The C-rich strand of telomere DNA in eukaryotes is tran- mentary to the template sequence of the telomerase RNA scribed into a long non-coding RNA called the telomeric re- (hTR), TERRA has also been shown to directly inhibit the peats containing RNA (TERRA) (1–3). Due to the repeated telomere repeat elongation activity of telomerase holoen- nature of the telomere DNA (TTAGGG deoxynucleotides zyme by binding to the template region of the hTR. Conse- repeats in vertebrates), the TERRA RNA sequence con- quently, a three-state model for the regulation of telomerase sists of UUAGGG nucleotide repeats. TERRA RNA has activity was proposed, where hnRNPA1 was propounded to been shown to localize at the telomere ends and play es- alleviate the TERRA mediated telomerase inhibition, by di- sential roles in telomere maintenance, heterochromatin for- rectly binding to and titrating away the TERRA RNA (22).
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