ACA Motif Research Articles

MazF-rolling circle amplification combined MALDI-TOF MS for site-specific detection of N6-methyladenosine RNA

N6-methyladenosine (m6A) is one of the most abundant chemical modifications in RNA and has vital significance in cellular processes and tumor development. However, the accurate analysis of site-specific m6A modification remains a challenge. In this work, a MazF endoribonuclease activated rolling circle amplification (MazF-RCA) combined MALDI-TOF MS assay is developed for the detection of site-specific m6A-RNA. MazF endoribonuclease can specifically cleave the ACA motif, leaving methylated (m6A)CA motif intact. The intact methylated RNA can then be amplified through rolling circle amplification, and the generated reporter oligonucleotides are detected by MALDI-TOF MS. The assay exhibits good quantification ability, presenting a wide linear range (100 fM to 10 nM) with the limit-of-detection lower than 100 fM. Additionally, the assay can accurately detect methylated RNA in the presence of large amount of non-methylated RNA with a relative abundance of methylated RNA down to 0.5%. The developed assay was further applied to detect m6A-RNA spiked in MCF-7 cell RNA extracts, with the recovery rates in the range of 90.64–106.93%. The present assay provides a novel platform for the analysis of site-specific m6A-RNA at high specificity and sensitivity, which can promote the study of RNA methylation in clinical and biomedical research.

Landscape of RNA pseudouridylation in archaeon Sulfolobus islandicus.

Pseudouridine, one of the most abundant RNA modifications, is synthesized by stand-alone or RNA-guided pseudouridine synthases. Here, we comprehensively mapped pseudouridines in rRNAs, tRNAs and small RNAs in the archaeon Sulfolobus islandicus and identified Cbf5-associated H/ACA RNAs. Through genetic deletion and in vitro modification assays, we determined the responsible enzymes for these modifications. The pseudouridylation machinery in S. islandicus consists of the stand-alone enzymes aPus7 and aPus10, and six H/ACA RNA-guided enzymes that account for all identified pseudouridines. These H/ACA RNAs guide the modification of all eleven sites in rRNAs, two sites in tRNAs, and two sites in CRISPR RNAs. One H/ACA RNA shows exceptional versatility by targeting eight different sites. aPus7 and aPus10 are responsible for modifying positions 13, 54 and 55 in tRNAs. We identified four atypical H/ACA RNAs that lack the lower stem and the ACA motif and confirmed their function both in vivo and in vitro. Intriguingly, atypical H/ACA RNAs can be modified by Cbf5 in a guide-independent manner. Our data provide the first global view of pseudouridylation in archaea and reveal unexpected structures, substrates, and activities of archaeal H/ACA RNPs.

Mapping single-nucleotide m6A by m6A-REF-seq.

The past few years have witnessed rapid progress in the field of RNA modifications. As the most prevailing modification on eukaryotic mRNA, m6A is characterized to play a vital role in various cellular activities. However, limitations of the detection method impede functional studies of m6A. Here we introduce m6A-REF-seq, a powerful and straightforward method to identify m6A at single-nucleotide resolution. m6A-REF-seq relies on the recognition of RNA endonuclease MazF towards m6A at the ACA motif, providing an orthogonal method independent of the m6A antibody being adopted by most of current methods. We describe a detailed protocol to perform m6A-REF-seq, including NGS library construction and sequencing data analysis. In particular, we describe an optimized assay to validate individual m6A sites identified by m6A-REF-seq, which can also be applied to detect any candidate m6A sites.

GcvB small RNA uses two distinct seed regions to regulate an extensive targetome.

GcvB small RNA is described as post-transcriptional regulator of 1-2% of all mRNAs in Escherichia coli and Salmonella Typhimurium. At least 24 GcvB:mRNA interactions have been validated in vivo, establishing the largest characterized sRNA targetome. By performing MS2-affinity purification coupled with RNA sequencing (MAPS) technology, we identified seven additional mRNAs negatively regulated by GcvB in E. coli. Contrary to the vast majority of previously known targets, which pair to the well-conserved GcvB R1 region, we validated four mRNAs targeted by GcvB R3 region. This indicates that base-pairing through R3 seed sequence seems relatively common. We also noticed unusual GcvB pairing sites in the coding sequence of two target mRNAs. One of these target mRNAs has a pairing site displaying a unique ACA motif, suggesting that GcvB could hijack a translational enhancer element. The second target mRNA is likely regulated via an active RNase E-mediated mRNA degradation mechanism. Remarkably, we confirmed the importance of the sRNA sponge SroC in the fine-tuning control of GcvB activity in function of growth conditions such as growth phase and nutrient availability.

Efficient RNA pseudouridylation by eukaryotic H/ACA ribonucleoproteins requires high affinity binding and correct positioning of guide RNA.

H/ACA ribonucleoproteins (H/ACA RNPs) are responsible for introducing many pseudouridines into RNAs, but are also involved in other cellular functions. Utilizing a purified and reconstituted yeast H/ACA RNP system that is active in pseudouridine formation under physiological conditions, we describe here the quantitative characterization of H/ACA RNP formation and function. This analysis reveals a surprisingly tight interaction of H/ACA guide RNA with the Cbf5p–Nop10p–Gar1p trimeric protein complex whereas Nhp2p binds comparably weakly to H/ACA guide RNA. Substrate RNA is bound to H/ACA RNPs with nanomolar affinity which correlates with the GC content in the guide-substrate RNA base pairing. Both Nhp2p and the conserved Box ACA element in guide RNA are required for efficient pseudouridine formation, but not for guide RNA or substrate RNA binding. These results suggest that Nhp2p and the Box ACA motif indirectly facilitate loading of the substrate RNA in the catalytic site of Cbf5p by correctly positioning the upper and lower parts of the H/ACA guide RNA on the H/ACA proteins. In summary, this study provides detailed insight into the molecular mechanism of H/ACA RNPs.

Translation enhancing ACA motifs and their silencing by a bacterial small regulatory RNA.

GcvB is an archetypal multi-target small RNA regulator of genes involved in amino acid uptake or metabolism in enteric bacteria. Included in the GcvB regulon is the yifK locus, encoding a conserved putative amino acid transporter. GcvB inhibits yifK mRNA translation by pairing with a sequence immediately upstream from the Shine-Dalgarno motif. Surprisingly, we found that some target sequence mutations that disrupt pairing, and thus were expected to relieve repression, actually lower yifK expression and cause it not to respond to GcvB variants carrying the corresponding compensatory changes. Work prompted by these observations revealed that the GcvB target sequence in yifK mRNA includes elements that stimulate translation initiation. Replacing each base of an ACA trinucleotide near the center of the target sequence, by any other base, caused yifK expression to decrease. Effects were additive, with some triple replacements causing up to a 90% reduction. The enhancer activity did not require the ACA motif to be strictly positioned relative to the Shine-Dalgarno sequence, nor did it depend on a particular spacing between the latter and the initiating AUG. The dppA mRNA, another GcvB target, contains four ACA motifs at the target site. Quite strikingly, replacement of all four ACAs by random trinucleotide sequences yielded variants showing over 100-fold reduction in expression, virtually inactivating the gene. Altogether, these data identify the ACA motif as a translation-enhancing module and show that GcvB's ability to antagonize the enhancer function in target mRNAs is quintessential to the regulatory effectiveness of this sRNA.

Significant Bias against the ACA Triplet in the tmRNA Sequence of Escherichia coli K-12

The toxin MazF in Escherichia coli cleaves single-stranded RNAs specifically at ACA sequences. MazF overexpression virtually eliminates all cellular mRNAs to completely block protein synthesis. However, protein synthesis can continue on an mRNA that is devoid of ACA triplets. The finding that ribosomal RNAs remain intact in the face of complete translation arrest suggested a purpose for such preservation. We therefore examined the sequences of all transcribed RNAs to determine if there was any statistically significant bias against ACA. While ACA motifs are absent from tmRNA, 4.5S RNA, and seven of the eight 5S rRNAs, statistical analysis revealed that only for tmRNA was the absence nonrandom. The introduction of single-strand ACAs makes tmRNA highly susceptible to MazF cleavage. Furthermore, analysis of tmRNA sequences from 442 bacteria showed that the discrimination against ACA in tmRNAs was seen mostly in enterobacteria. We propose that the unusual bias against ACA in tmRNA may have coevolved with the acquisition of MazF.

18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryote-specific 18S sequences

The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non-coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of ‘orphan' H/ACA RNAs participate in pre-rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 ‘orphan' H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre-rRNA processing, two evolutionarily conserved sequence elements in the 3′-hairpin of snR30 base-pair with short pre-rRNA sequences located in the eukaryote-specific internal region of 18S rRNA. The newly discovered snR30-18S base-pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3′-hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein-binding site.

A Conserved WD40 Protein Binds the Cajal Body Localization Signal of scaRNP Particles

Small Cajal body (CB)-specific RNPs (scaRNPs) function in posttranscriptional modification of small nuclear (sn)RNAs. An RNA element, the CAB box, facilitates CB localization of H/ACA scaRNPs. Using a related element in Drosophila C/D scaRNAs, we purified a fly WD40 repeat protein that UV crosslinks to RNA in a C/D CAB box-dependent manner and associates with C/D and mixed domain C/D-H/ACA scaRNAs. Its human homolog, WDR79, associates with C/D, H/ACA, and mixed domain scaRNAs, as well as with telomerase RNA. WDR79's binding to human H/ACA and mixed domain scaRNAs is CAB box dependent, and its association with mixed domain RNAs also requires the ACA motif, arguing for additional interactions of WDR79 with H/ACA core proteins. We demonstrate a requirement for WDR79 binding in the CB localization of a scaRNA. This and other recent reports establish WDR79 as a central player in the localization and processing of nuclear RNPs.

Structure and Function of the Smallest Vertebrate Telomerase RNA from Teleost Fish

Telomerase extends chromosome ends by copying a short template sequence within its intrinsic RNA component. Telomerase RNA (TR) from different groups of species varies dramatically in sequence and size. We report here the bioinformatic identification, secondary structure comparison, and functional analysis of the smallest known vertebrate TRs from five teleost fishes. The teleost TRs (312-348 nucleotides) are significantly smaller than the cartilaginous fish TRs (478-559 nucleotides) and tetrapod TRs. This remarkable length reduction of teleost fish TRs correlates positively with the genome size, reflecting an unusual structural plasticity of TR during evolution. The teleost TR consists of a compact three-domain structure, lacking most of the sequences in regions that are variable in other vertebrate TR structures. The medaka and fugu TRs, when assembled with their telomerase reverse transcriptase (TERT) protein counterparts, reconstituted active and processive telomerase enzymes. Titration analysis of individual RNA domains suggests that the efficient assembly of the telomerase complex is influenced more by the telomerase reverse transcriptase (TERT) binding of the CR4-CR5 domain than the pseudoknot domain of TR. The remarkably small teleost fish TR further expands our understanding about the evolutionary divergence of vertebrate TR.

Reconstitution of archaeal H/ACA small ribonucleoprotein complexes active in pseudouridylation

Pseudouridine (Ψ) are frequently modified residues in RNA. In Eukarya, their formation is catalyzed by enzymes or by ribonucleoprotein complexes (RNPs) containing H/ACA snoRNAs. H/ACA sRNA and putative ORFs for H/ACA sRNP proteins (L7Ae, aCBF5, aNOP10 and aGAR1) were found in Archaea. Here, by using Pyrococcus abyssi recombinant proteins and an in vitro transcribed P.abyssi H/ACA sRNA, we obtained the first complete in vitro reconstitution of an active H/ACA RNP. Both L7Ae and the aCBF5 RNA:Ψ synthase bind directly the sRNA; aCBF5 also interacts directly and independently with aNOP10 and aGAR1. Presence of aCBF5, aNOP10 and a U residue at the pseudouridylation site in the target RNA are required for RNA target recruitment. In agreement, we found that the aCBF5–aNOP10 pair is the minimal set of proteins needed for the formation of a particle active for pseudouridylation. However, particles more efficient in targeted pseudouridylation can be formed with the addition of proteins L7Ae and/or aGAR1. Although necessary for optimal activity, the conserved ACA motif in the sRNA was found to be not essential.

Characterization of three novel snoRNAs from Schizosaccharomyces pombe implies the structural and functional diversity of box H/ACA snoRNA family

Site-specific pseudouridylations of rRNAs and snRNAs are directed by small guide RNAs that usually possess conserved H and ACA motifs and the consensus secondary structure consisting of hairpin-hinge-hairpin-tail. In this study, through screening cDNA library, three novel box H/ACA snoRNAs, termed SP12, SP16 and SP20, have been identified from fisson yeast Schizosacchromyces pombe. Each of the small RNAs possesses an ACA-like motif and exhibits secondary structures containing two or three large hairpinlike domains. Although the box H is noncanonical, SP20 can be potentially folded into a typical box H/ACA snoRNA-like structure and is predicted to guide two pseudouridylations, U1208 and U1053, on 18S rRNA. In contrast, SP12 and SP16 display different structures from SP20 and have no antisense elements to rRNAs or snRNAs. Obviously, they are new members of orphan guide snoRNAs whose functions remain elusive. The genes encoded for SP12 and SP16 are both located in long intergenic regions between protein-coding genes. A post-transcriptional processing for SP16 precursor was demonstrated. Interestingly, SP20 gene is nested inside a very hypothetical ORF, but transcribed in a reverse sense. Northern and RT-PCR analyses could not detect the transcript from the hypothetical ORF, and therefore exclude the expression of the spurious gene. This is the first example that a genetic locus predicted for protein-coding gene actually encodes a small non-messenger RNA in S. pombe.

A small nucleolar guide RNA functions both in 2'-O-ribose methylation and pseudouridylation of the U5 spliceosomal RNA.

In eukaryotes, two distinct classes of small nucleolar RNAs (snoRNAs), namely the fibrillarin-associated box C/D snoRNAs and the Gar1p-associated box H/ACA snoRNAs, direct the site-specific 2'-O-ribose methylation and pseudouridylation of ribosomal RNAs (rRNAs), respectively. We have identified a novel evolutionarily conserved snoRNA, called U85, which possesses the box elements of both classes of snoRNAs and associates with both fibrillarin and Gar1p. In vitro and in vivo pseudouridylation and 2'-O-methylation experiments provide evidence that the U85 snoRNA directs 2'-O-methylation of the C45 and pseudouridylation of the U46 residues in the invariant loop 1 of the human U5 spliceosomal RNA. The U85 is the first example of a snoRNA that directs modification of an RNA polymerase II-transcribed spliceosomal RNA and that functions both in RNA pseudouridylation and 2'-O-methylation.

Mutational analysis of upstream sequences required for transcriptional activation of the Klebsiella pneumoniae nifH promoter.

Upstream sequences of the Klebsiella pneumoniae nifH promoter were mutagenised and activation of the mutated promoters by the nif-specific transcriptional activator protein NifA examined in vivo. Of the sixteen mutations analysed, only those within the nifH upstream activator sequence (UAS), characterised by a TGT-N10-ACA motif, influenced nifH promoter activity. Mutations altering the two-fold rotational symmetry of the UAS or the spacing between the TGT and ACA motifs reduced promoter activity, consistent with the UAS functioning as a NifA binding site. The bases flanking the TGT-ACA motif of the UAS also appear to influence activation by NifA. Substituting the nifH UAS with a binding site for the transcriptional activator NtrC resulted in improved NtrC-dependent activation of the nifH promoter demonstrating that the activator specificity of the nifH promoter is dependent upon the presence of the appropriate upstream sequences to which the activator binds.