Transcription factors induce differential splicing of duplicated ribosomal protein genes during meiosis.

In baker's yeast, genes encoding ribosomal proteins often exist as duplicate pairs, typically with one 'major' paralog highly expressed and a 'minor' less expressed paralog that undergoes controlled expression through reduced splicing efficiency. In this study, we investigate the regulatory mechanisms controlling splicing of the minor paralog of the uS4 protein gene (RPS9A), demonstrating that its splicing is repressed during vegetative growth but upregulated during meiosis. This differential splicing of RPS9A is mediated by two transcription factors, Rim101 and Taf14.

Canada's contributions to RNA research: past, present, and future perspectives.

The field of RNA research has provided profound insights into the basic mechanisms modulating the function and adaption of biological systems. RNA has also been at the center stage in the development of transformative biotechnological and medical applications, perhaps most notably was the advent of mRNA vaccines that were critical in helping humanity through the Covid-19 pandemic. Unbeknownst to many, Canada boasts a diverse community of RNA scientists, spanning multiple disciplines and locations, whose cutting-edge research has established a rich track record of contributions across various aspects of RNA science over many decades.

Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar KD.

Fluorogenic RNA aptamer tags with high affinity enable RNA purification and imaging. The G-quadruplex (G4) based Mango (M) series of aptamers were selected to bind a thiazole orange based (TO1-Biotin) ligand. Using a chemical biology and reselection approach, we have produced a MII.

Turn-on RNA Mango Beacons for -acting fluorogenic nucleic acid detection.

The Mango I and II RNA aptamers have been widely used in vivo and in vitro as genetically encodable fluorogenic markers that undergo large increases in fluorescence upon binding to their ligand, TO1-Biotin. However, while studying nucleic acid sequences, it is often desirable to have -acting probes that induce fluorescence upon binding to a target sequence. Here, we rationally design three types of light-up RNA Mango Beacons based on a minimized Mango core that induces fluorescence upon binding to a target RNA strand.

Harmonizing the growing fluorogenic RNA aptamer toolbox for RNA detection and imaging.

The field of fluorogenic RNA aptamers is a burgeoning research area that aims to address the lack of naturally fluorescent RNA molecules for RNA detection and imaging. These small RNA tags bind to their fluorogenic ligands resulting in significant fluorescent enhancement, leading to a molar brightness comparable to or exceeding that of fluorescent proteins. In the past decade, multiple light-up RNA aptamer systems have been isolated that bind to a broad range of ligands involving several distinct mechanisms of fluorogenicity.

6S RNA complexed to RNA polymerase maintains product RNA synthesis at low cellular ATP levels by initiation with noncanonical initiator nucleotides.

The 6S RNA is an RNA polymerase (RNAP) inhibitor that competes with σ-dependent DNA promoters for binding to RNAP holoenzyme (RNAP:σ). The 6S RNA when bound is then used as a template to synthesize a short product RNA (pRNA; usually 13-nt-long). This pRNA changes the 6S RNA structure, triggering the 6S RNA:pRNA complex to release and allowing DNA-dependent housekeeping gene expression to resume.

Single-Molecule RNA Imaging Using Mango II Arrays.

In recent years, fluorogenic RNA aptamers, such as Spinach, Broccoli, Corn, Mango, Coral, and Pepper have gathered traction as an efficient alternative labeling strategy for background-free imaging of cellular RNAs. However, their application has been somewhat limited by relatively inefficient folding and fluorescent stability. With the recent advent of novel RNA-Mango variants which are improved in both fluorescence intensity and folding stability in tandem arrays, it is now possible to image RNAs with single-molecule sensitivity.

Processive RNA polymerization and promoter recognition in an RNA World.

Early life is thought to have required the self-replication of RNA by RNA replicases. However, how such replicases evolved and subsequently enabled gene expression remains largely unexplored. We engineered and selected a holopolymerase ribozyme that uses a sigma factor-like specificity primer to first recognize an RNA promoter sequence and then, in a second step, rearrange to a processive elongation form.

Microchip RT-PCR Detection of Nasopharyngeal SARS-CoV-2 Samples.

Fast, accurate, and reliable diagnostic tests are critical for controlling the spread of the coronavirus disease 2019 (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The current gold standard for testing is real-time PCR; however, during the current pandemic, supplies of testing kits and reagents have been limited. We report the validation of a rapid (30 minutes), user-friendly, and accurate microchip real-time PCR assay for detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts.

RNA Peach and Mango: Orthogonal two-color fluorogenic aptamers distinguish nearly identical ligands.

Two-channel fluorogenic RNA aptamer-based imaging is currently challenging. While we have previously characterized the Mango series of aptamers that bind tightly and specifically to the green fluorophore TO1-Biotin, the next aim was to identify an effective fluorogenic aptamer partner for two-color imaging. A competitive in vitro selection for TO3-Biotin binding aptamers was performed resulting in the Peach I and II aptamers.

Fluorogenic aptamers resolve the flexibility of RNA junctions using orientation-dependent FRET.

To further understand the transcriptome, new tools capable of measuring folding, interactions, and localization of RNA are needed. Although Förster resonance energy transfer (FRET) is an angle- and distance-dependent phenomenon, the majority of FRET measurements have been used to report distances, by assuming rotationally averaged donor-acceptor pairs. Angle-dependent FRET measurements have proven challenging for nucleic acids due to the difficulties in incorporating fluorophores rigidly into local substructures in a biocompatible manner.

Structure-Guided Engineering of the Homodimeric Mango-IV Fluorescence Turn-on Aptamer Yields an RNA FRET Pair.

Fluorescent RNA aptamers have been used in cells as biosensor reporters and tags for tracking transcripts. Recently, combined SELEX and microfluidic fluorescence sorting yielded three aptamers that activate fluorescence of TO1-Biotin: Mango-II, Mango-III, and Mango-IV. Of these, Mango-IV was best at imaging RNAs in both fixed and live mammalian cells.

Live cell imaging of single RNA molecules with fluorogenic Mango II arrays.

RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding.

RNA detection with high specificity and sensitivity using nested fluorogenic Mango NASBA.

There is a pressing need for nucleic acid-based assays that are capable of rapidly and reliably detecting pathogenic organisms. Many of the techniques available for the detection of pathogenic RNA possess one or more limiting factors that make the detection of low-copy RNA challenging. Although RT-PCR is the most commonly used method for detecting pathogen-related RNA, it requires expensive thermocycling equipment and is comparatively slow.

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method.

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19-22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest.

Structure and functional reselection of the Mango-III fluorogenic RNA aptamer.

Several turn-on RNA aptamers that activate small-molecule fluorophores have been selected in vitro. Among these, the ~30 nucleotide Mango-III is notable because it binds the thiazole orange derivative TO1-Biotin with high affinity and fluoresces brightly (quantum yield 0.55).

Genome-wide discovery of somatic regulatory variants in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL.

Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness.

Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ∼1 nM affinity and enhances its fluorescence by >1500-fold, was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity.

Purification of RNA Mango Tagged Native RNA-protein Complexes from Cellular Extracts Using TO1-Desthiobiotin Fluorophore Ligand.

A native purification strategy using RNA Mango for RNA based purification of RNA-protein complexes is described. The RNA Mango aptamer is first genetically engineered into the RNA of interest. RNA Mango containing complexes obtained from cleared cellular native extracts are then immobilized onto TO1-Desthiobiotin saturated streptavidin agarose beads.

High-throughput detection of RNA processing in bacteria.

Background: Understanding the RNA processing of an organism's transcriptome is an essential but challenging step in understanding its biology. Here we investigate with unprecedented detail the transcriptome of Pseudomonas aeruginosa PAO1, a medically important and innately multi-drug resistant bacterium. We systematically mapped RNA cleavage and dephosphorylation sites that result in 5'-monophosphate terminated RNA (pRNA) using monophosphate RNA-Seq (pRNA-Seq).

Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers.

Transitioning to DNA genomes in an RNA world.

The unexpected ability of an RNA polymerase ribozyme to copy RNA into DNA has ramifications for understanding how DNA genomes evolved.

Ribonucleoprotein purification and characterization using RNA Mango.

The characterization of RNA-protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin.

Structural basis for high-affinity fluorophore binding and activation by RNA Mango.

Genetically encoded fluorescent protein tags have revolutionized proteome studies, whereas the lack of intrinsically fluorescent RNAs has hindered transcriptome exploration. Among several RNA-fluorophore complexes that potentially address this problem, RNA Mango has an exceptionally high affinity for its thiazole orange (TO)-derived fluorophore, TO1-Biotin (K ∼3 nM), and, in complex with related ligands, it is one of the most redshifted fluorescent macromolecular tags known. To elucidate how this small aptamer exhibits such properties, which make it well suited for studying low-copy cellular RNAs, we determined its 1.

Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers.

The effective tracking and purification of biological RNAs and RNA protein complexes is currently challenging. One promising strategy to simultaneously address both of these problems is to develop high-affinity RNA aptamers against taggable small molecule fluorophores. RNA Mango is a 39-nucleotide, parallel-stranded G-quadruplex RNA aptamer motif that binds with nanomolar affinity to a set of thiazole orange (TO1) derivatives while simultaneously inducing a 10-fold increase in fluorescence.