RNA

Ribosomal proteins, because of their RNA-binding capacity, may engage various cellular RNAs and fulfill nonribosomal roles. Previously, we and others described the intergenic regulation mediated by splicing of RPL22 paralogs in Saccharomyces cerevisiae. Here, we prepared a panel of RPL22A/B intronic mutants with respect to their RNAfold-predicted features and analyzed their properties. We tested the splicing and Rpl22-intron interaction using an intron-containing reporter and a three-hybrid yeast system, respectively. We found that the splicing of RPL22 introns can be inhibited by stabilizing a predicted stem as part of a particular type of conformation (I structure). Stabilizing the formation of an alternate stem (P structure) led to a permissive outcome of splicing. Intriguingly, the regulatory capacity of the main stem loop of the I structure was dependent on the rest of the intronic structure. Rpl22 enhanced splicing inhibition in WT and several of the mutants, which we interpret as stabilization of the I structure by protein binding. Mutagenesis identified both the main and alternative 5ss and additional stem loops as part of the regulatory mechanism. The inhibitory conformation of the intron did not prevent recognition of the 5ss and branch point, but rather stalled splicing at a later stage, before the first catalytic step. We concluded that the structural ensemble of the RPL22 pre-mRNA behaves as an allosteric switch that responds to [Rpl22].

1Circular RNAs have garnered considerable interest, as they have been implicated in numerous biological processes and diseases. Through their stability, they are often considered promising biomarker candidates or therapeutic targets. Due to the lack of a poly(A) tail, circRNAs are best detected in total RNA-seq data after depleting ribosomal RNA. However, we observe that the application of circRNA detection in the vastly more ubiquitous poly(A)-enriched RNA-seq data still occurs. In this study, we systematically compare the detection of circRNAs in two matched poly(A) and ribosomal RNA-depleted data sets. Our results indicate that the comparably few circRNAs detected in poly(A) data are likely false positives. In addition, we demonstrate that the quality of sample processing, as measured by the fraction of ribosomal reads, significantly affects the sensitivity of circRNA detection, leading to a bias in downstream analysis. Our findings establish best practices for circRNA research: total RNA sequencing with effective rRNA depletion is the preferred approach for accurate circRNA profiling, whereas poly(A)-enriched data are unsuitable for comprehensive detection. Employing multiple circRNA detection tools and prioritizing back-splice junctions identified by several algorithms enhances confidence in the selection of candidates. These recommendations, validated across diverse datasets and tissue types, provide generalizable principles for robust circRNA analysis. Key PointsO_LIRibosomal RNA contamination substantially impairs the accuracy of circRNA detection. This technical confounding factor has thus far received limited attention in the field. C_LIO_LITool agreement for circRNA calls is moderate in total RNA-seq but essentially absent in poly(A)-enriched RNA-seq data, underscoring the importance of using multiple tools for circRNA detection. C_LIO_LIBack-splice junctions detected in poly(A)-enriched RNA-seq data are predominantly tool-specific artifacts rather than genuine circRNAs, challenging the validity of circRNA identification in poly(A)-enriched datasets. C_LI

i-Motifs are non-canonical nucleic acid secondary structures formed in sequences rich in cytosine. Previous work has shown that DNA i-motifs may form at neutral pH and provided evidence to suggest they influence biological functions. RNA i-motifs are less stable than DNA i-motifs, so more questions surround the biological relevance of these structures in RNA. Using biophysical methods, we found that increasing cytosine tract lengths resulted in increased thermal stability but not pH stability. In ensemble solution experiments RNA i-motifs appear globally unfolded at neutral pH. However, single molecule experiments revealed that 1% of RNA i-motifs remain folded in solution. This has implications for the potential of formation of RNA i-motifs in cells.

The Arabidopsis thaliana La1 (AtLa1) protein is a member of the genuine La family of RNA biogenesis proteins, which are structurally similar to the La-resembling protein 7 (LARP7) family. LARP7 proteins participate in the biogenesis of the telomerase ribonucleoprotein complex in model systems, but are absent in plants. We show that AtLa1 binds to telomerase RNA in a manner reminiscent of the Tetrahymena LARP7 protein p65. Classical in vitro methods and microscale thermophoresis (MST) were used to specify the molecular structures involved in this multi-surface interaction. AtLa1 also enhances the binding of TR to the telomerase reverse transcriptase RNA binding domain. We therefore propose that biogenesis of telomerase RNA in plants and ciliates is achieved by a similar pathway, differing in the employment of genuine La or LARP7-like proteins, respectively. We also report that the domain of unknown function (DUF3223, DeCL) found in the AtLa1 protein binding partner, Domino, is an RNA binding domain with modest TR-binding capacity. This domain is also found in plant and ciliate proteins, including plant polymerases IV/V and the Tetrahymena La protein Mlp1. Together, these suggest that RNA biogenesis pathways in plants and ciliates have a conserved evolutionary relationship, with parallels between their La proteins.

Regulation of RNA pools allows for adaptation to changing environments and stress, which is especially important in pathogenic bacteria such as Mycobacterium tuberculosis. RNA degradation is a significant contributor to RNA abundance, and Ribonuclease (RNase) E has a rate-limiting role in degradation of a majority of mycobacterial transcripts. However, many open questions remain about the RNA substrate requirements and specificities for efficient cleavage by mycobacterial RNase E. Here, using both Mycolicibacterium smegmatis and M. tuberculosis RNase E, we demonstrate that this enzyme is only active on substrates with a minimum length of approximately 27 nt. Furthermore, we show that mycobacterial RNase E prefers substrates with 5 monophosphates rather than 5 triphosphates, and that the positions of cleavage events within substrates are dictated by both sequence and distance from the RNA ends. Our results also suggest that RNase E may be affected by product inhibition. Finally, we show that M. smegmatis RNase E behaves similarly to M. tuberculosis RNase E, validating the use of this model organism for RNA degradation studies.

Like other cells, parasitic and other trypanosomatids sense and regulate Zn2+ transport, but the mechanisms involved remained unknown. Here we identify a trypanosome RNA-binding protein which specifically eliminates ZIP3 Zn2+-transporter mRNA in Zn2+-replete conditions. We first demonstrated that Trypanosoma brucei ZIP3 mRNA abundance is subject to 3-untranslated region (3-UTR) and Zn2+-dependent negative control. A genome-wide RNA interference library screen, using a reporter associated with the ZIP3 3-UTR, identified Tb927.11.9510 as a candidate Zn2+-sensor. We name this protein Zinc Nuclear Knuckles 1 (ZNK1) since it localises to the nucleus and contains several Zn2+-knuckle motifs. ZNK1 is conserved among trypanosomatids, and a PIN-domain suggests a ribonuclease-based mechanism. We validate ZNK1 as a ZIP3 3-UTR dependent negative regulator and identify a GU-repeat motif in the ZIP3 3-UTR that is predictive of negative control by ZNK1. We use Cas9-editing to knockout ZNK1, and RNA-seq to assess the consequences, revealing highly specific accumulation of ZIP3 transcripts in znk1-null cells. We conclude that ZNK1 senses Zn2+-abundance and eliminates ZIP3 mRNA in a Zn2+-dependent manner. We suggest that trypanosomatid ZNK1 is an RNA-specific zinc finger nuclease that binds ZIP3 3-UTRs and degrades ZIP3 mRNA only when the tandem sensor modules are coordinated with Zn2+. Key pointsO_LITrypanosome Zinc Nuclear Knuckles 1 (ZNK1) is a zinc-sensor that eliminates zinc transporter mRNA. C_LIO_LIZNK1 negative control operates via the transporter mRNA 3-untranslated region. C_LIO_LIThe findings indicate that trypanosomatid ZNK1 is a conserved RNA-specific zinc finger nuclease. C_LI

Escherichia coli YicC enzyme is the founding member of a family of endoribonucleases that is encoded in virtually all bacterial species. Previous structural studies revealed that this ribonuclease binds RNA by a novel mechanism in which the hexameric apoprotein presents an open channel that undergoes a large rotation upon RNA binding and clamps down on the RNA. The current study follows up on these findings by examining the cleavage of various oligonucleotide substrates designed to probe recognition elements required for YicC binding and cleavage. A 26-nucleotide RNA oligomer (oligo), with a KD in the low micromolar range, was the standard to which numerous oligos with altered sequence were compared. In vitro RNase assays and fluorescence anisotropy binding measurements indicated that the preferred substrates for YicC were relatively small RNAs that contain some secondary structure. Larger RNAs or highly structured RNAs were less-than-optimal substrates. Similarly, RyhB RNA, a [~]90-nucleotide, iron-responsive RNA of E. coli, which has been described as a target of YicC binding and/or cleavage, was a poor YicC substrate in our assays. These results suggest that the native substrates for YicC-family members are very small RNAs or RNA fragments derived from larger RNAs.

BackgroundSmall non-coding RNAs (sncRNAs) play central roles in post-transcriptional gene regulation. In addition to canonical microRNAs (miRNAs), fragments derived from vault RNAs (vtRNAs), called small vault RNAs (svtRNAs), have been reported in human cells. However, the absence of a standardized annotation framework has hindered their systematic detection, quantification, and comparison across small RNA sequencing (small RNA-seq) studies. MethodsWe developed an expression-based annotation strategy to identify svtRNAs from human small RNA-seq datasets. Using FlaiMapper followed by structure and expression-based filtering, we generated two annotation sets: a stringent "miRNA-like" set enriched in Argonaute-associated datasets, and (ii) a broader "Total" set derived from total small RNA-seq libraries under relaxed structural constraints. We explored the expression of the annotated svtRNAs across the different datasets analyzed: multiple normal and tumor-derived human cell lines, including Argonaute immunoprecipitation datasets. ResultsWe identified a repertoire of svtRNAs that are detected across independent datasets and, in several cases, reach abundance levels comparable to canonical miRNAs. Several highly abundant svtRNAs correspond to molecules with experimental validation from prior studies, supporting the robustness of our annotation strategy. Importantly, the same "dominant" (in terms of gene expression) svtRNAs emerged independently from Argonaute-associated and total small RNA datasets, supporting the idea of enzymatically consistent, reproducible svtRNA processing. We further identified svtRNAs derived from distinct vtRNA precursors that could share identical seed sequences, suggesting the possibility of svtRNA families with potential miRNA-like regulatory properties. We provide a standardized annotation that enables reproducible svtRNA quantification. ConclusionsOur study establishes a comprehensive expression-based annotation resource for human svtRNAs. By enabling their systematic detection and reproducible quantification, we show that svtRNAs appear to represent an abundant component of the human small RNA landscape.

Pre-mRNA splicing is an essential step in eukaryotic gene expression during which spliceosomes remove introns from nascent RNAs while ligating the adjacent exons. Spliceosomes are cellular nanomachines composed of five small nuclear (snRNA) components and dozens of proteins, most of which are highly conserved. Despite the high conservation of many splicing factors between S. cerevisiae and H. sapiens, several protein components of the S. cerevisiae spliceosome are not essential for growth under normal laboratory conditions. This is particularly surprising for nonessential factors whose conserved domains contact the spliceosomes catalytic core. Uncovering a function for these splicing factors can be challenging since they are not required for viability, may engage in functionally redundant interactions, and may display only weak phenotypes in the absence of secondary mutations in other spliceosome components. One such nonessential factor is the Cwc15 protein. Cwc15s highly conserved N-terminus directly contacts the U2/U6 di-snRNA within the spliceosome catalytic core; yet its precise role in splicing has not been defined in any organism. In this work, we use molecular genetics in S. cerevisiae combined with splicing reporter assays to study Cwc15p function. We propose that Cwc15p not only promotes active site stability during 5 splice site cleavage but also impacts structural transitions into and out of this spliceosome conformation. This function may be critical for splicing in S. cerevisiae under nonoptimal conditions, facilitating use of weak or alternate splice sites, and could have implications for proofreading of spliceosome active site formation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/713263v1_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@b296c5org.highwire.dtl.DTLVardef@c87b91org.highwire.dtl.DTLVardef@287011org.highwire.dtl.DTLVardef@d59741_HPS_FORMAT_FIGEXP M_FIG C_FIG Article SummaryPre-mRNA splicing is carried out by large macromolecular machines called spliceosomes which are composed of several snRNAs and dozens of proteins. Despite decades of study, the functions of many splicing factors such as S. cerevisiae Cwc15p remain unknown. Cwc15p is highly conserved among eukaryotes and directly contacts the spliceosome catalytic core. Here, we have used genetic and splicing reporter assays to study the function of Cwc15p during splicing in vivo. We propose that Cwc15p both stabilizes the spliceosome active site during 5 splice site cleavage and impacts remodeling of that site.

RNA thermometers (RNATs) are temperature-responsive structures in 5' untranslated regions (UTRs) of bacterial messenger RNA (mRNA) that control translation by modulating ribosome access. The Listeria monocytogenes prfA RNAT represses translation of PrfA (positive regulatory factor A), the master virulence regulator, at ambient temperature and activates it near the human host temperature ([~]37 {degrees}C) by modulating ribosome binding site (RBS) accessibility. However, the prfA RNAT shares no homology with known RNAT classes, and its unfolding mechanism remains unclear. Here, we used analytical ultracentrifugation and single-molecule kinetic analysis of RNA transient structure (SiM-KARTS) to map prfA RNAT unfolding. SiM-KARTS analysis demonstrates that thermal opening occurs predominantly at the RBS, while the upper helix of the RNAT hairpin remains largely folded at 37 {degrees}C. The RBS binding kinetics increases with temperature in parallel with translation output, establishing a quantitative link between structural unfolding and function. Mutations in the upper helix impair thermal regulation, indicating that this region tunes switching even as it stays structured at host temperature. Together, these data reveal a hierarchical unfolding pathway in which initial RBS opening triggers activation, whereas the upper helix remotely tunes temperature sensitivity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/717274v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@a95daborg.highwire.dtl.DTLVardef@144bc42org.highwire.dtl.DTLVardef@1a39bf6org.highwire.dtl.DTLVardef@545e60_HPS_FORMAT_FIGEXP M_FIG C_FIG

A morphospace is an abstract space of theoretically possible biological traits, shapes, or property values. It is interesting to explore which parts of a morphospace life occupies, as compared to those parts which could be occupied, but are not. Comparing random and natural non-coding (nc) RNA secondary structures is an established approach to studying morphospace occupation for RNA structures. Most earlier studies have focused on the minimum free energy (MFE) structure, while relatively few have looked at the Boltzmann distribution, describing the ensemble of energetically suboptimal RNA folds. These suboptimal structures may have important roles and functions, and hence should be examined carefully. Here we compare random and natural ncRNA in terms of their Boltzmann distributions, finding that natural RNA tend to have very similar profiles to random RNA, with the main difference being that natural RNA are slightly more energetically stable, except for very short sequences (20 to 30 nucleotides) which tend to be slightly less stable. We infer that natural ncRNA occupy similar parts of the morphospace that random RNA do, indicating that the biophysics of the genotype-phenotype map largely determines the ensemble properties of ncRNA.

Triple-negative breast cancer (TNBC) remains one of the most aggressive breast cancer subtypes and lacks durable targeted therapies. Dysregulation of cell-cycle control, particularly through CDK4/6 signaling, is a defining feature of TNBC biology (Garrido-Castro et al., 2019). Extracts of Moringa oleifera have repeatedly been shown to induce G1-phase arrest in breast cancer models, yet the molecular basis of this phenotype remains unclear (Al-Asmari et al., 2015) (Gaffar et al., 2019). Emerging work on cross-kingdom regulation has raised the possibility that plant-derived microRNAs may, under specific conditions, interact with mammalian transcripts (Zhang et al., 2012) (Chin et al., 2016). Sequence shuffling for the negative control was performed with set.seed(42) to ensure reproducibility. Additional visualisations (nucleotide alignment and thermodynamic analyses) were generated using Python 3 (matplotlib v3.7). Here, we performed a high-stringency computational screen of conserved Moringa microRNAs against 30 genes implicated in TNBC pathogenesis using local sequence alignment. We identify a predicted high-affinity interaction between mol-miR156 and the human CDK4 3' untranslated region (3'UTR), characterized by an uninterrupted 12-nucleotide complementary motif that exceeds canonical mammalian microRNA seed requirements. These findings support the hypothesis that conserved plant microRNAs may exhibit latent structural compatibility with oncogenic human transcripts. While physiological delivery and functional repression are not demonstrated here, this work establishes a molecular framework for future experimental investigation into cross-kingdom RNA interactions relevant to cancer cell-cycle regulation. Impact StatementA high-stringency computational screen identifies latent molecular compatibility between a conserved plant microRNA and the human CDK4 oncogene, establishing a testable framework for cross-kingdom RNA interference in triple-negative breast cancer.

The ability to access, search, and analyse large collections of RNA molecules together with their secondary structure and evolutionary context is essential for comparative and phylogeny-driven studies. Although RNA secondary structure is known to be more conserved than primary sequence, no existing resource systematically associates individual RNA molecules with curated phylogenetic classifications. Here, we introduce PhyloRNA, a curated meta-database that provides large-scale access to RNA secondary structures collected from public resources or derived from experimentally resolved 3D structures. PhyloRNA allows users to search, select, and download extensive sets of RNA molecules in multiple textual formats, each entry being explicitly linked to phylogenetic annotations derived from five curated taxonomy systems. In addition to taxonomic information, each RNA molecule is accompanied by a rich set of descriptors, including pseudoknot order, genus, and three levels of structural abstraction--Core, Core Plus, and Shape--which facilitate comparative analyses across sets of molecules. PhyloRNA is publicly available at https://bdslab.unicam.it/phylorna/ and is regularly updated to incorporate newly available data and revised taxonomic annotations.

Motivation RNA structure plays a central role in how transcripts function, but inferring it reliably remains difficult, especially when pseudoknots need to be part of the prediction. Chemical probing experiments provide additional signals, yet these signals do not directly identify base pairing partners. RNA proximity ligation provides direct evidence of base pairing, but balancing this evidence with pseudoknot prediction accuracy and scalability of structure prediction for long sequences remains challenging. Results We present CPLfold, a fast and flexible RNA folding method that combines thermodynamic modeling with chimeric evidence from RNA cross-linking and ligation experiments, while naturally supporting pseudoknots. CPLfold scales to long sequences and recovers more accurate global structures and long-range interactions than existing approaches across multiple benchmarks such as COMRADES and IRIS. By tuning two simple trade-off parameters (, {beta}) the method allows flexibility in the level of incorporating chimeric evidence and asserting pseudoknots. Availability and Implementation Source code and scripts are available at https://github.com/Vicky-0256/CPLfold. ContactK.Wang-72@sms.ed.ac.uk

Since the discovery of viral Internal Ribosome Entry Sites (IRESes), researchers have sought to find similar elements in mammalian host genes, termed "cellular IRESes". However, the plasmid systems used to measure cellular IRES activity are vulnerable to false positives due to promoter activity in candidate IRESes. Orthogonal methods are needed to validate putative IRESes while carefully avoiding artifacts known to cause false positives. Recently, Koch et al. proposed approaches for studying IRESes, primarily circular RNA-generating plasmids, and for validating mRNA transcripts using smFISH and qRT-PCR. Here, we demonstrate confounding variables and artifacts in each of these approaches that can lead to inappropriate conclusions about potential cellular IRES activity. We show the back-splicing circRNA plasmid creates linear mRNA artifacts associated with false-positive IRES signals. Using orthogonal, gold-standard assays validated with viral IRESes, we find putative cellular IRESes reported using the back-splicing plasmid have no IRES activity. Furthermore, we demonstrate that smFISH and qRT-PCR can misidentify nuclear non-coding RNAs as mRNAs and we validate a single molecule sequencing assay for identifying genuine mRNA 5 ends. Our work establishes reliable methods for robust transcript annotation and IRES studies that avoid documented artifacts arising from bicistronic and back-splicing circRNA plasmid reporters.

The correct assembly of ribosomes is essential for viability and faithful gene expression. In eukaryotic cells, the pre-40S and pre-60S ribosomal subunits are largely pre-assembled in the nucleolus before they are exported to the cytoplasm for final maturation. Although most ribosomal proteins of the large subunit are loaded onto pre-60S particles in the early nucleolar steps, a few, including eL24, are loaded in the cytoplasm. eL24 is thought to recruit the zinc-finger protein Rei1 (ZNF622 in humans). In yeast, Rei1 has a paralog, Reh1. While we and others have previously shown that Rei1 facilitates the removal of Arx1, Rei1 and Reh1 appear to have an additional unknown function. To identify this function, we first examined the protein composition of pre-60S subunits isolated from rei1{Delta} reh1{Delta} mutant cells and found that these subunits were specifically defective for eL24. However, the absence of eL24 did not impair Rei1 binding to pre-60S. Moreover, overexpression of eL24 suppressed the growth defect of the double mutant. As an alternative approach to understanding the function of Rei1 and Reh1, we screened for bypass suppressors of the growth defect of rei1{Delta} reh1{Delta} cells. We identified mutations in the genes coding for ribosomal protein uL3, the GTPase Lsg1 and the protein phosphatase Ppq1. Importantly, these suppressors all partially reversed the eL24 loading defect of rei1{Delta} reh1{Delta} cells. Based on these results, we propose a revised order of cytoplasmic assembly events where Rei1 and Reh1 facilitate the recruitment of eL24 to the pre-60S particle.

Biomolecular condensates are central to subcellular compartmentalization. Although many condensates contain and regulate RNA, research has primarily focused on protein interactions. Here, we investigate RNA-protein interactions underlying cell cycle-regulating condensates in the multinucleate fungus Ashbya gossypii. These condensates form through interactions between G1 cyclin mRNA CLN3 and RNA-binding protein Whi3, which was predicted by homology to recognize a five-nucleotide motif repeated within the transcript. Natural variation in motif number in Ashbya strains led us to hypothesize that binding site valence may influence condensate properties. Using unbiased binding assays, we determined the preference of Ashbya Whi3 protein for specific primary RNA sequence and mutated individual Whi3-binding sites within CLN3 mRNA. Mutants exhibited distinct condensate properties despite having the same valence in terms of binding site number. Mutations altered the saturation concentration (Csat) and dense phase concentration of RNA and protein in cell-free reconstitution experiments. A subset of mutants, showed reduced number of condensates and deregulation of the cell cycle in cells. We also find that enhanced availability of single-stranded RNA can compensate for loss of binding sites. Together, these data indicate that differences in RNA protein binding-site context and not simply valence plays a critical role in determining condensate properties.

Accurate detection of RNA splice variants is often hindered when transcripts lack large distinguishable exonic regions, making conventional PCR strategies challenging. We developed a simple melting temperature (Tm)-guided exon-exon junction (EEJ) RT-PCR method to enable variant-specific detection under these conditions. Uni-directional primers spanning exon-exon junctions were designed so that approximately each half anneals to adjacent exons. The Tm of each half-site was set >7{degrees}C below the annealing temperature, preventing stable binding to individual exons and enforcing junction-dependent amplification. The method was evaluated using HTRA1-AS1 long noncoding RNA variants that share overlapping exon sequences but differ in splice connectivity. HTRA1-AS1 comprises five variants, only one with a large distinguishable exon. Tm-guided EEJ primers robustly discriminated the remaining four variants. After optimization, amplification yielded sharp, single bands with minimal cross-reactivity. Compared with conventional designs, this approach reduced heteroduplex and heteroquadruplex formation, improving band clarity. Sanger sequencing confirmed junction specificity, and the method performed well in multiplex settings. Overall, Tm-guided EEJ RT-PCR is a cost-effective, high-resolution approach for detecting RNA variants lacking easily distinguishable exonic regions, readily compatible with standard RT-PCR and qPCR workflows.

Long non-coding RNAs (lncRNAs) account for a major proportion of the transcriptional output in complex organismal genomes. Their emergence as auxiliary regulators of gene expression as well as their roles in metastasis and cancer progression has put them in the limelight. LncRNAs perform multitudes of functions and often moonlight as regulators, scaffolds and guides. Most lncRNAs are cell and tissue specific and can act as markers for diseases as well as targets for therapeutic interventions. LncRNAs are also known to make use of higher order structures such as G-quadruplexes (G4) to facilitate complex functions and interactions. THAP9-antisense1 (AS1) is a lncRNA coding gene (recently annotated by Ensembl) that codes for 12 lncRNA transcripts and has been implicated in many disease pathologies like gastric cancer, spontaneous neutrophil apoptosis, hepatocellular carcinoma, and the progression of oesophageal cancer. It is the antisense gene pair of the THAP9 gene ( a transposase derived gene) with which it shares a promoter. THAP9-AS1 has been reported to be dysregulated during stress and several cancers. However, the exact role of the lncRNA is not well understood. Bioinformatics driven strategies are used to identify putative quadruplex forming sequences (PQSs) within the lncRNA THAP9-AS1. The identified PQSs are further validated using biophysical, spectroscopic and molecular biology driven techniques. The importance of each G-tract in the formation of a particular RNA G-quadruplex (rG4) is studied via the investigation of several deletion mutants. The findings demonstrate the rG4 forming potential of the identified PQSs within THAP9-AS1.

Eukaryotic gene expression is a multi-layered process influenced by multiple factors. One of them is the secondary structure of precursor mRNAs that can impact various aspects of their processing including alternative splicing (AS). Here, we report the functional characterization of the conserved RNA structural element DEAD that is located in DEAD-box RNA helicase (DRH) genes from land plants and serves as a sensor for RNA helicase activity by controlling AS. In Arabidopsis thaliana, it is found in DRH1 and its closest paralog, regulating usage of an alternative splice site as part of a negative feedback loop. Accordingly, opening of the structure shifts splicing towards non-coding variants, thereby balancing transcript and protein levels. Interestingly, the system is specific to DRH1 and its paralog and does not react to related helicases, which is at least partially conferred by the disordered and RGG/RG motif-containing C-terminus of DRH1. The importance of DEAD is underlined by the observation that releasing this attenuation mechanism causes massive changes in AS - mainly intron retention and exon skipping - and gene expression and results in a severe stress phenotype. Thus, DEAD provides a critical buffering mechanism to fine-tune helicase levels and their global impact on RNA structure-responsive gene expression.