Visualizing a protonated RNA state that modulates microRNA-21 maturation

Abstract

MicroRNAs are evolutionarily conserved small, non-coding RNAs that regulate diverse biological processes. Due to their essential regulatory roles, microRNA biogenesis is tightly regulated, where protein factors are often found to interact with specific primary and precursor microRNAs for regulation. Here, using NMR relaxation dispersion spectroscopy and mutagenesis, we reveal that the precursor of oncogenic microRNA-21 exists as a pH-dependent ensemble that spontaneously reshuffles the secondary structure of the entire apical stem-loop region, including the Dicer cleavage site. We show that the alternative excited conformation transiently sequesters the bulged adenine into a non-canonical protonated A + -G mismatch, conferring a two-fold enhancement in Dicer processing over its ground conformational state. These results indicate that microRNA maturation efficiency may be encoded in the intrinsic dynamic ensemble of primary and precursor microRNAs, providing potential means of regulating microRNA biogenesis in response to environmental and cellular stimuli. Page 3 MicroRNAs (miRNAs) are highly conserved, small noncoding RNAs that regulate more than 60% of protein coding genes at the post-transcriptional level 1-5 . Most miRNAs are initially transcribed by RNA polymerase II as introns of protein-coding genes or from independent coding genes into long primary transcripts (pri-miRNAs) that feature 5'-end 7-methylguanosine caps and 3'-end poly-A tails 6,7 . In the canonical biogenesis pathway, pri-miRNAs are subsequently processed into ~70 nucleotide precursor hairpins (pre-miRNAs) by the Microprocessor complex, consisting of one RNase III family enzyme, Drosha, and two DiGeorge critical region 8 proteins (DGCR8) 8,9 . Pre-miRNAs are then exported from the nucleus to the cytoplasm by Exportin-5 (Ref. 10) and further processed into ~20 base-pair miRNA/miRNA* duplexes by another RNase III family enzyme, Dicer, in complex with transactivation-responsive RNA binding protein (TRBP) 11,12 . The resulting single-stranded mature miRNA is incorporated into the miRNA-inducing silencing complex (miRISC), which regulates protein expression by repressing translation, promoting deadenylation, and/or cleaving target mRNA 13 .Due to their essential regulatory roles, miRNA biogenesis is tightly regulated to ensure proper gene expression 3-5 , and abnormal miRNA regulation has often been associated with cancer, neurological disorders, cardiovascular diseases and others 14,15 .Remarkably, despite sharing the same set of enzymes in the canonical biogenesis pathway, individual miRNAs exhibit cell-type and cell-state specific expressions. Even those clustered on the same primary transcript can be differentially processed in a tissue-specific manner 3-5 . Over the past decade, it has been shown that specific sequences and structures of primary and precursor miRNAs can be recognized by processing machineries and protein factors for regulation [16][17][18][19][20][21][22][23][24][25][26] . For example, pri-miRNAs

Aromatic 19F–13C TROSY—[19F, 13C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA

“…20 kD) ( Figure 1d). We further incorporated a [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] Our initial efforts focused on the production of the [5-19 F, 5-13 C]-uridine phosphoramidite 7.B ased on our recently published synthetic access to a5-13 C-uridine building block [9] we included af luorination step using the commercially available fluorination reagent Selectfluor TM .T he synthetic route starting from [5-13 C]-uracil is shown (Scheme 1). The compound 1 was produced from [2-13 C]-bromoacetic acid, potassium cyanide and urea as previously described.…”

“…Schwalbe and coworkers used the latter method to probe the effect of all 2-F-Al abeling in the 73 nt guanine sensing riboswitch from bacillus subtilis. [3a] Only four resonances of sixteen could be assigned illustrating that in larger RNAs resonance assignment issues arise.Thus,anexpansion of the 19 Fstable isotope labeling scheme to yield a 19 F- 13 C-spin topology,such as a [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 5-13 C]-labeling pattern in pyrimidines,would be desirable to evade resonance overlap issues in larger RNAs by adding asecond 13 Cdimension. Theapplication of 19 F-NMR to study high molecular weight systems is also hampered by the fast transverse relaxation by the chemical shift anisotropy (CSA) mechanism.…”

“…In ar ecent work this issue was addressed by introducing aromatic 19 F- 13 C-spin topologies in proteins and DNA, in which the cancellation of the dipole-dipole interaction and the chemical shift anisotropy relaxation mechanisms lead to avery favorable NMR spectroscopic behavior in aTROSY type experiment. [6] Here,wereport on the synthetic access to [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 5-13 C]-uridine (U) and -cytidine (C) phosphoramidites for solid phase RNAsynthesis.W edemonstrate the favorable spectroscopic behavior of the [5-19 F, 5-13 C]pyrimidines in two model RNAs:the 61 nt human hepatitis B virus encapsidation signal epsilon (hHBV e)a nd the 59 nt pre-microRNA21. [7] Thefeasibility of the 13 C/ 19 Flabeling by solid phase RNAsynthesis is shown and used for aresonance assignment protocol based on [5-19 F, 5-13 C]/[5-19 F]-uridine labeling schemes.F or the 61 nt hHBV e RNAw eo bserved afavorable TROSY effect and compared the performance of [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 13 C]-U labels with [6-1 H, 6-13 C]-U labels.T he novel stable isotope (SI) labeling scheme will facilitate NMR studies of large RNAs and high molecular weight RNA protein complexes as it was recently demonstrated for high molecular weight proteins systems.…”

Aromatic 19F–13C TROSY—[19F, 13C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA

“…[13] This is corroborated by as tudy of Zhang and co-workers,i nw hich ap H-dependent excited state in the apical stem loop of pre-miRNA2 1w as identified. [14] We picked the non-coding pre-miRNA2115 as ap romising target for our labeling approach in view of an application of the 19 F/ 13 C-pyrimidines in in vivo NMR to follow the fate of pre-miRNAs in cells.W ea lso tested the combined U/C-19 F/ 13 C-labeling. Them odular composition of the pre-miRNA2 1w as confirmed by an almost identical chemical shift pattern of the apical stem loop with [5-19 F, 5- (Figure 5c and d).…”

“…Forschungsartikel phosphoramidites 7 and 11 were available RNAs with sizes between 30 and 60 nts were synthesized. First, the 30 nt HIV TAR-2 with six uridines and the 61 nt hHBV e RNAw ith eighteen uridines were synthesized and six and five [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 5-13 C]-U labels were incorporated in each RNA, respectively. We obtained high quality 19 F- 13 CT ROSY spectra within as hort time on aP rodigy TCI probe with the proton coil tuned to the 19 Fr esonance frequency for 0.5 mm RNA samples.T he additional 13 Cd imension allowed to resolve the resonance overlap,w hich was earlier observed for U23, U25 and U31 in the 1D-19 Fs pectrum.…”