pH dependence of C•A, G•A and A•A mismatches in the stem of precursor microRNA-31

Kotar, Anita; Ma, Sicong; Keane, Sarah C.

doi:10.1101/2021.10.25.465784

Cited by 3 publications

(6 citation statements)

References 88 publications

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“…We previously reported chemical shift assignments for two fragments, BottomA and BottomB. [44] We completed chemical shift assignments for two additional oligo fragments, TopA ( Fig. S3 ) and Top ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Base pair mismatches are a common feature within the helical stem of precursor microRNAs [44] . Increasing the length of the pre-miR helical stem by including additional base paired sequences is detrimental for Dicer processing [14, 49] .…”

Section: Resultsmentioning

confidence: 99%

“…We previously investigated the pH-dependence of the C18•A54 mismatch and found that A54 is partially protonated at physiological pH, suggesting that these bases can form a C•A + base pair near neutral pH [44] . We were therefore interested in testing if mutations that replaced the mismatch with a canonical U-A or C-G base pair (C18U and A54G, respectively) affected the processing by Dicer ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Structure of pre-miR-31 reveals an active role in Dicer processing

Kotar

Grote

et al. 2023

Preprint

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As an essential post-transcriptional regulator of gene expression, microRNA (miR) levels must be strictly maintained. The biogenesis of many, but not all, miRs is mediated by trans-acting protein partners through a variety of mechanisms, including remodeling of the RNA structure. miR-31 functions as an oncogene in numerous cancers and interestingly, its biogenesis is not known to be regulated by protein binding partners. Therefore, the intrinsic structural properties of pre-miR-31 can provide a mechanism by which its biogenesis is regulated. We determined the solution structure of the precursor element of miR-31 (pre-miR-31) to investigate the role of distinct structural elements in regulating Dicer processing. We found that the presence or absence of mismatches within the helical stem do not strongly influence Dicer processing of the pre-miR. However, both the apical loop size and structure at the Dicing site are key elements for discrimination by Dicer. Interestingly, our NMR-derived structure reveals the presence of a triplet of base pairs that link the Dicer cleavage site and the apical loop. Mutational analysis in this region suggests that the stability of the junction region strongly influence both Dicer binding and processing. Our results enrich our understanding of the active role that RNA structure plays in regulating Dicer processing which has direct implications for control of gene expression.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Structure of pre-miR-31 reveals an active role in Dicer processing

Kotar

Grote

et al. 2023

Preprint

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“…Protonation is a fundamental chemical property and the smallest modification on RNA and DNA molecules. 44,45 The single stranded RNA and DNA nucleobases are typically uncharged, the intrinsic p K a values for adenine and cytosine are far from neutrality. 46,47 However, in some instances, the adenosine p K a shifts towards neutrality, enabling their participation in non-canonical base pairings.…”

Section: Resultsmentioning

confidence: 99%

Solution structure of NPSL2, a regulatory element in the oncomiR-1 RNA

Liu

Hall

Keane

2022

Preprint

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The miR-17~92a polycistron, also known as oncomiR-1, is commonly overexpressed in multiple cancers and has several oncogenic properties. OncomiR-1 encodes six constituent microRNAs (miRs), each enzymatically processed with different efficiencies. However, the structural mechanism that regulates this differential processing remains unclear. Chemical probing of oncomiR-1 revealed that the Drosha cleavage sites of pri-miR-92a are sequestered in a four-way junction. NPSL2, an independent stem loop element, is positioned just upstream of pri-miR-92a and sequesters a crucial part of the sequence that constitutes the basal helix of pri-miR-92a. Disruption of the NPSL2 hairpin structure could promote the formation of a pri-miR-92a structure that is primed for processing by Drosha. Thus, NPSL2 is predicted to function as a structural switch, regulating pri-miR-92a processing. Here, we determined the solution structure of NPSL2 using solution NMR spectroscopy. This is the first high-solution structure of an oncomiR-1 element. NPSL2 adopts a hairpin structure with a large, but highly structured, apical and internal loops. The 10-bp apical loop contains a pH-sensitive A+·C mismatch. Additionally, several adenosines within the apical and internal loops have elevated pKa values. The protonation of these adenosines can stabilize the NPSL2 structure through electrostatic interactions. Our study provides fundamental insights into the secondary and tertiary structure of an important RNA hairpin proposed to regulate miR biogenesis.

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“…The temperature was moved to the target value, and the system was allowed to equilibrate for one minute before a measurement was taken. Thermal melting data was fit to a two-state model with sloping baselines 48 ( Equation 1 ) in Prism 9. All RNAs tested exhibited reversible thermal melting profiles.…”

Section: Methodsmentioning

confidence: 99%

Rare Ribosomal RNA Sequences from Archaea Stabilize the Bacterial Ribosome

Nissley

Penev

Watson

et al. 2022

Preprint

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Ribosomes serve as the universally conserved translators of the genetic code into proteins and must support life across temperatures ranging from below freezing to above the boiling point of water. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Peptide bond formation by the ribosome requires correct positioning of the 3′-end of the aminoacylated tRNA (aa-tRNA) substrate, which is aided by an RNA hairpin in the ribosomal RNA (rRNA) of the large subunit, termed the A loop. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3′-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post- transcriptional modification of the A loop. Additionally, these mutations do not exert a dominant negative effect on cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation in the peptidyl transferase center of the archaeal ribosome that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

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pH dependence of C•A, G•A and A•A mismatches in the stem of precursor microRNA-31

Cited by 3 publications

References 88 publications

Structure of pre-miR-31 reveals an active role in Dicer processing

Structure of pre-miR-31 reveals an active role in Dicer processing

Solution structure of NPSL2, a regulatory element in the oncomiR-1 RNA

Rare Ribosomal RNA Sequences from Archaea Stabilize the Bacterial Ribosome

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