Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing.

RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

Genome wide analysis of 3' UTR sequence elements and proteins regulating mRNA stability during maternal-to-zygotic transition in zebrafish.

Posttranscriptional regulation plays a crucial role in shaping gene expression. During the maternal-to-zygotic transition (MZT), thousands of maternal transcripts are regulated. However, how different cis-elements and trans-factors are integrated to determine mRNA stability remains poorly understood. Here, we show that most transcripts are under combinatorial regulation by multiple decay pathways during zebrafish MZT. By using a massively parallel reporter assay, we identified cis-regulatory sequences in the 3' UTR, including U-rich motifs that are associated with increased mRNA stability. In contrast, miR-430 target sequences, UAUUUAUU AU-rich elements (ARE), CCUC, and CUGC elements emerged as destabilizing motifs, with miR-430 and AREs causing mRNA deadenylation upon genome activation. We identified trans-factors by profiling RNA-protein interactions and found that poly(U)-binding proteins are preferentially associated with 3' UTR sequences and stabilizing motifs. We show that this activity is antagonized by C-rich motifs and correlated with protein binding. Finally, we integrated these regulatory motifs into a machine learning model that predicts reporter mRNA stability in vivo.

CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.

CRISPR-Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observed that guanine enrichment and adenine depletion increased sgRNA stability and activity, whereas differential sgRNA loading, nucleosome positioning and Cas9 off-target binding were not major determinants. We also identified sgRNAs truncated by one or two nucleotides and containing 5' mismatches as efficient alternatives to canonical sgRNAs. On the basis of these results, we created a predictive sgRNA-scoring algorithm, CRISPRscan, that effectively captures the sequence features affecting the activity of CRISPR-Cas9 in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos 3' UTR led to the generation of maternal-zygotic mutants, as well as increased viability and decreased somatic mutations. These results identify determinants that influence Cas9 activity and provide a framework for the design of highly efficient sgRNAs for genome targeting in vivo.

Small antisense oligonucleotides against G-quadruplexes: specific mRNA translational switches.

G-quadruplexes (G4) are intricate RNA structures found throughout the transcriptome. Because they are associated with a variety of biological cellular mechanisms, these fascinating structural motifs are seen as potential therapeutic targets against many diseases. While screening of chemical compounds specific to G4 motifs has yielded interesting results, no single compound successfully discriminates between G4 motifs based on nucleotide sequences alone. This level of specificity is best attained using antisense oligonucleotides (ASO). Indeed, oligonucleotide-based strategies are already used to modulate DNA G4 folding in vitro. Here, we report that, in human cells, the use of short ASO to promote and inhibit RNA G4 folding affects the translation of specific mRNAs, including one from the 5'UTR of the H2AFY gene, a histone variant associated with cellular differentiation and cancer. These results suggest that the relatively high specificity of ASO-based strategies holds significant potential for applications aimed at modulating G4-motif folding.

New scoring system to identify RNA G-quadruplex folding.

G-quadruplexes (G4s) are non-canonical structures involved in many important cellular processes. To date, the prediction of potential G-quadruplex structures (PG4s) has been based almost exclusively on the sequence of interest agreeing with the algorithm Gx-N-1-7-Gx-N1-7-Gx-N1-7-Gx (where x ≥ 3 and N = A, U, G or C). However, many sequences agreeing with this algorithm do not form G4s and are considered false-positive predictions. Here we show the RNA PG4 candidate in the 3'-untranslated region (UTR) of the TTYH1 gene to be one such false positive. Specifically, G4 folding was observed to be inhibited by the presence of multiple-cytosine tracks, located in the candidate's genomic context, that adopted a Watson-Crick base-paired structure. Clearly, the neighbouring sequences of a PG4 may influence its folding. The secondary structure of 12 PG4 motifs along with either 15 or 50 nucleotides of their upstream and downstream genomic contexts were evaluated by in-line probing. Data permitted the development of a scoring system for the prediction of PG4s taking into account the effect of the neighbouring sequences. The accuracy of this scoring system was assessed by probing 14 other novel PG4 candidates retrieved in human 5'-UTRs. This new scoring system can be used, in combination with the standard algorithm, to better predict the folding of RNA G4s.

Exploring mRNA 3'-UTR G-quadruplexes: evidence of roles in both alternative polyadenylation and mRNA shortening.

Guanine-rich RNA sequences can fold into non-canonical, four stranded helical structures called G-quadruplexes that have been shown to be widely distributed within the mammalian transcriptome, as well as being key regulatory elements in various biological mechanisms. That said, their role within the 3'-untranslated region (UTR) of mRNA remains to be elucidated and appreciated. A bioinformatic analysis of the 3'-UTRs of mRNAs revealed enrichment in G-quadruplexes. To shed light on the role(s) of these structures, those found in the LRP5 and FXR1 genes were characterized both in vitro and in cellulo. The 3'-UTR G-quadruplexes were found to increase the efficiencies of alternative polyadenylation sites, leading to the expression of shorter transcripts and to possess the ability to interfere with the miRNA regulatory network of a specific mRNA. Clearly, G-quadruplexes located in the 3'-UTRs of mRNAs are cis-regulatory elements that have a significant impact on gene expression.

In-line probing of RNA G-quadruplexes.

Although the majority of the initial G-quadruplex studies were performed on DNA molecules, there currently exists a rapidly growing interest in the investigation of those formed in RNA molecules that possess high potential of acting as gene expression regulatory elements. Indeed, G-quadruplexes found in the 5'-untranslated regions of mRNAs have been reported to be widespread within the human transcriptome and to act as general translational repressors. In addition to translation regulation, several other mRNA maturation steps and events, including mRNA splicing, polyadenylation and localization, have been shown to be influenced by the presence of these RNA G-quadruplexes. Bioinformatic approaches have identified thousands of potential RNA G-quadruplex sequences in the human transcriptome. Clearly there is a need for the development of rapid, simple and informative techniques and methodologies with which the ability of these sequences, and of any potential new regulatory elements, to fold into G-quadruplexes in vitro can be examined. This report describes an integrated methodology for monitoring RNA G-quadruplexes formation that combines bioinformatic algorithms, secondary structure prediction, in-line probing with semi-quantification analysis and structural representation software. The power of this approach is illustrated, step-by-step, with the determination of the structure adopted by a potential G-quadruplex sequence found in the 5'-untranslated region of the cAMP responsive element modulator (CREM) mRNA. The results unambiguously show that the CREM sequence folds into a G-quadruplex structure in the presence of a physiological concentration of potassium ions. This in-line probing-based method is easy to use, robust, reproducible and informative in the study of RNA G-quadruplex formation.

5'-UTR G-quadruplex structures acting as translational repressors.

Given that greater than 90% of the human genome is expressed, it is logical to assume that post-transcriptional regulatory mechanisms must be the primary means of controlling the flow of information from mRNA to protein. This report describes a robust approach that includes in silico, in vitro and in cellulo experiments permitting an in-depth evaluation of the impact of G-quadruplexes as translational repressors. Sequences including potential G-quadruplexes were selected within nine distinct genes encoding proteins involved in various biological processes. Their abilities to fold into G-quadruplex structures in vitro were evaluated using circular dichroism, thermal denaturation and the novel use of in-line probing. Six sequences were observed to fold into G-quadruplex structures in vitro, all of which exhibited translational inhibition in cellulo when linked to a reporter gene. Sequence analysis, direct mutagenesis and subsequent experiments were performed in order to define the rules governing the folding of G-quadruplexes. In addition, the impact of single-nucleotide polymorphism was shown to be important in the formation of G-quadruplexes located within the 5'-untranslated region of an mRNA. In light of these results, clearly the 5'-UTR G-quadruplexes represent a class of translational repressors that is broadly distributed in the cell.

Potassium ions modulate a G-quadruplex-ribozyme's activity.

Hepatitis delta virus ribozyme folds into a tightly packed tertiary structure. However, unlike other ribozymes, it does not appear to be able to follow alternative folding pathways. Molecular engineering of the hepatitis delta virus ribozyme led to the development of a ribozyme possessing an endoribonuclease activity that is under the control of a G-quadruplex structure (i.e., a G-quartzyme). This latter species represents an entirely new class of ribozyme. Mutants of this ribozyme were then generated in order to shed light on the modulation of the cleavage activity caused by the presence of the G-quadruplex structure. Kinetic characterization of the G-quartzyme was performed under various single turnover conditions. It was found to be active only in the presence of potassium cations that act as counter ions in the positioning of the four coplanar guanines that form the building block of the G-quadruplex structure. The G-quartzyme behaves as an allosteric ribozyme, with the potassium cations acting as positive effectors with a Hill coefficient of 2.9 +/- 0.2. The conformation transition caused by the presence of the potassium ions is supported by enzymatic and chemical probing of both the inactive (off) and active (on) structures. This study shows that it is possible to interfere with the tight structure of the hepatitis delta virus ribozyme by adding an unusual, stable structure. To our knowledge, the G-quartzyme is the sole ribozyme that exhibits a monovalent cation-dependent activity.

Modulating RNA structure and catalysis: lessons from small cleaving ribozymes.

RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today's knowledge in the field.

A Functional Non-coding RNA Is Produced from xbp-1 mRNA.

The xbp-1 mRNA encodes the XBP-1 transcription factor, a critical part of the unfolded protein response. Here we report that an RNA fragment produced from xbp-1 mRNA cleavage is a biologically active non-coding RNA (ncRNA) essential for axon regeneration in Caenorhabditis elegans. We show that the xbp-1 ncRNA acts independently of the protein-coding function of the xbp-1 transcript as part of a dual output xbp-1 mRNA stress response axis. Structural analysis indicates that the function of the xbp-1 ncRNA depends on a single RNA stem; this stem forms only in the cleaved xbp-1 ncRNA fragment. Disruption of this stem abolishes the non-coding, but not the coding, function of the endogenous xbp-1 transcript. Thus, cleavage of the xbp-1 mRNA bifurcates it into a coding and a non-coding pathway; modulation of the two pathways may allow neurons to fine-tune their response to injury and other stresses.

A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme.

A bioinformatic covariation analysis of a collection of 119 novel variants of the antigenomic, self-cleaving hepatitis delta virus (HDV) RNA motif supported the formation of all of the Watson-Crick base pairs (bp) of the catalytic centre except the C19-G81 pair located at the bottom of the P2 stem. In fact, a novel Watson-Crick bp between C19 and G80 is suggested by the data. Both chemical and enzymatic probing demonstrated that initially the C19-G81 pair is formed in the ribozyme (Rz), but upon substrate (S) binding and the formation of the P1.1 pseudoknot C19 switches its base-pairing partner from G81 to G80. As a result of this finding, the secondary structure of this ribozyme has been redrawn. The formation of the C19-G80 bp results in a J4/2 junction composed of four nucleotides, similar to that seen in the genomic counterpart, thereby increasing the similarities between these two catalytic RNAs. Additional mutagenesis, cleavage activity and probing experiments yield an original characterization of the structural features involving the residues of the J4/2 junction.

Brd4 and P300 Confer Transcriptional Competency during Zygotic Genome Activation.

The awakening of the genome after fertilization is a cornerstone of animal development. However, the mechanisms that activate the silent genome after fertilization are poorly understood. Here, we show that transcriptional competency is regulated by Brd4- and P300-dependent histone acetylation in zebrafish. Live imaging of transcription revealed that genome activation, beginning at the miR-430 locus, is gradual and stochastic. We show that genome activation does not require slowdown of the cell cycle and is regulated through the translation of maternally inherited mRNAs. Among these, the enhancer regulators P300 and Brd4 can prematurely activate transcription and restore transcriptional competency when maternal mRNA translation is blocked, whereas inhibition of histone acetylation blocks genome activation. We conclude that P300 and Brd4 are sufficient to trigger genome-wide transcriptional competency by regulating histone acetylation on the first zygotic genes in zebrafish. This mechanism is critical for initiating zygotic development and developmental reprogramming.

In vitro selection and characterization of RNA aptamers binding thyroxine hormone.

RNA possesses the ability to bind a wide repertoire of small molecules. Some of these binding interactions have been shown to be of primary importance in molecular biology. For example, several classes of mRNA domains, collectively referred to as riboswitches, have been shown to serve as RNA genetic control elements that sense the concentrations of specific metabolites (i.e. acting as direct sensors of chemical compounds). However, to date no RNA species binding a hormone has been reported. Here, we report that the use of an appropriate SELEX (systematic evolution of ligands by exponential enrichment) strategy results in the isolation of thyroxine-specific aptamers. Further biochemical characterization of these aptamers, including mutational studies, the use of transcripts with site-specific modified nucleotides, nuclease and chemical probing, binding-shift assays and CD, demonstrated that these RNA structures included a G-rich motif, reminiscent of a guanine quadruplex structure, adjacent to a helical region. The presence of the thyroxine appeared to be essential for the formation of the structural motif's scaffold. Moreover, the binding is shown to be specific to thyroxine (T4) and tri-iodothyronine (T3), the active forms of the hormone, whereas other inactive derivatives, including thyronine (T0), do not support complex formation. These results suggest that this aptamer specifically binds to the iodine moieties of the thyroxine, a previously unreported ability for an RNA molecule.

Analyses of mRNA structure dynamics identify embryonic gene regulatory programs.

RNA folding plays a crucial role in RNA function. However, knowledge of the global structure of the transcriptome is limited to cellular systems at steady state, thus hindering the understanding of RNA structure dynamics during biological transitions and how it influences gene function. Here, we characterized mRNA structure dynamics during zebrafish development. We observed that on a global level, translation guides structure rather than structure guiding translation. We detected a decrease in structure in translated regions and identified the ribosome as a major remodeler of RNA structure in vivo. In contrast, we found that 3' untranslated regions (UTRs) form highly folded structures in vivo, which can affect gene expression by modulating microRNA activity. Furthermore, dynamic 3'-UTR structures contain RNA-decay elements, such as the regulatory elements in nanog and ccna1, two genes encoding key maternal factors orchestrating the maternal-to-zygotic transition. These results reveal a central role of RNA structure dynamics in gene regulatory programs.