Identification of leader-trailer helices of precursor ribosomal RNA in all phyla of bacteria and archaea.

Ribosomal RNAs are transcribed as part of larger precursor molecules. In Escherichia coli, complementary RNA segments flank each rRNA and form long leader-trailer (LT) helices, which are crucial for subunit biogenesis in the cell. A previous study of 15 representative species suggested that most but not all prokaryotes contain LT helices. Here, we use a combination of in silico folding and covariation methods to identify and characterize LT helices in 4464 bacterial and 260 archaeal organisms. Our results suggest that LT helices are present in all phyla, including Deinococcota, which had previously been suspected to lack LT helices. In very few organisms, our pipeline failed to detect LT helices for both 16S and 23S rRNA. However, a closer case-by-case look revealed that LT helices are indeed present but escaped initial detection. Over 3600 secondary structure models, many well supported by nucleotide covariation, were generated. These structures show a high degree of diversity. Yet, all exhibit extensive base-pairing between the leader and trailer strands, in line with a common and essential function.

The U1-70K and SRSF1 interaction is modulated by phosphorylation during the early stages of spliceosome assembly.

In eukaryotes, pre-mRNA splicing is vital for RNA processing and orchestrated by the spliceosome, whose assembly starts with the interaction between U1-70K and SR proteins. Despite the significance of the U1-70K/SR interaction, the dynamic nature of the complex and the challenges in obtaining soluble U1-70K have impeded a comprehensive understanding of the interaction at the structural level for decades. We overcome the U1-70K solubility issues, enabling us to characterize the interaction between U1-70K and SRSF1, a representative SR protein. We unveil specific interactions: phosphorylated SRSF1 RS with U1-70K BAD1, and SRSF1 RRM1 with U1-70K RRM. The RS/BAD1 interaction plays a dominant role, whereas the interaction between the RRM domains further enhances the stability of the U1-70K/SRSF1 complex. The RRM interaction involves the C-terminal extension of U1-70K RRM and the conserved acid patches on SRSF1 RRM1 that is involved in SRSF1 phase separation. Our circular dichroism spectra reveal that BAD1 adapts an α-helical conformation and RS is intrinsically disordered. Intriguingly, BAD1 undergoes a conformation switch from α-helix to ß-strand and random coil upon RS binding. In addition to the regulatory mechanism via SRSF1 phosphorylation, the U1-70K/SRSF1 interaction is also regulated by U1-70K BAD1 phosphorylation. We find that U1-70K phosphorylation inhibits the U1-70K and SRSF1 interaction. Our structural findings are validated through in vitro splicing assays and in-cell saturated domain scanning using the CRISPR method, providing new insights into the intricate regulatory mechanisms of pre-mRNA splicing.

Principles of miRNA/miRNA* function in plant MIRNA processing.

MicroRNAs (miRNAs) are essential regulators of gene expression, defined by their unique biogenesis, which requires the precise excision of the small RNA from an imperfect fold-back precursor. Unlike their animal counterparts, plant miRNA precursors exhibit variations in sizes and shapes. Plant MIRNAs can undergo processing in a base-to-loop or loop-to-base direction, with DICER-LIKE1 (DCL1) releasing the miRNA after two cuts (two-step MIRNAs) or more (sequential MIRNAs). In this study, we demonstrate the critical role of the miRNA/miRNA* duplex region in the processing of miRNA precursors. We observed that endogenous MIRNAs frequently experience suboptimal processing in vivo due to mismatches in the miRNA/miRNA* duplex, a key region that fine-tunes miRNA levels. Enhancing the interaction energy of the miRNA/miRNA* duplex in two-step MIRNAs results in a substantial increase in miRNA levels. Conversely, sequential MIRNAs display distinct and specific requirements for the miRNA/miRNA* duplexes along their foldback structure. Our work establishes a connection between the miRNA/miRNA* structure and precursor processing mechanisms. Furthermore, we reveal a link between the biological function of miRNAs and the processing mechanism of their precursors with the evolution of plant miRNA/miRNA* duplex structures.

Short 2'-O-methyl/LNA oligomers as highly-selective inhibitors of miRNA production in vitro and in vivo.

MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that members of the same miRNA family with high sequence similarity might have different roles and that this functional divergence might be rooted in their precursors' sequence. Current knock-down strategies such as antisense oligonucleotides (ASOs) or miRNA sponges cannot distinguish between identical or near identical miRNAs originating from different precursors to allow exploring unique functions of these miRNAs. We here develop a novel strategy based on short 2'-OMe/LNA-modified oligonucleotides to selectively target specific precursor molecules and ablate the production of individual members of miRNA families in vitro and in vivo. Leveraging the highly conserved Xenopus miR-181a family as proof-of-concept, we demonstrate that 2'-OMe/LNA-ASOs targeting the apical region of pre-miRNAs achieve precursor-selective inhibition of mature miRNA-5p production. Furthermore, we extend the applicability of our approach to the human miR-16 family, illustrating its universality in targeting precursors generating identical miRNAs. Overall, our strategy enables efficient manipulation of miRNA expression, offering a powerful tool to dissect the functions of identical or highly similar miRNAs derived from different precursors within miRNA families.

Molecular basis of human poly(A) polymerase recruitment by mPSF.

3' end processing of most eukaryotic precursor-mRNAs (pre-mRNAs) is a crucial cotranscriptional process that generally involves the cleavage and polyadenylation of the precursor transcripts. Within the human 3' end processing machinery, the four-subunit mammalian polyadenylation specificity factor (mPSF) recognizes the polyadenylation signal (PAS) in the pre-mRNA and recruits the poly(A) polymerase α (PAPOA) to it. To shed light on the molecular mechanisms of PAPOA recruitment to mPSF, we used a combination of cryogenic-electron microscopy (cryo-EM) single-particle analysis, computational structure prediction, and in vitro biochemistry to reveal an intricate interaction network. A short linear motif in the mPSF subunit FIP1 interacts with the structured core of human PAPOA, with a binding mode that is evolutionarily conserved from yeast to human. In higher eukaryotes, however, PAPOA contains a conserved C-terminal motif that can interact intramolecularly with the same residues of the PAPOA structured core used to bind FIP1. Interestingly, using biochemical assay and cryo-EM structural analysis, we found that the PAPOA C-terminal motif can also directly interact with mPSF at the subunit CPSF160. These results show that PAPOA recruitment to mPSF is mediated by two distinct intermolecular connections and further suggest the presence of mutually exclusive interactions in the regulation of 3' end processing.

Recent developments, opportunities, and challenges in the study of mRNA pseudouridylation.

Pseudouridine is an abundant mRNA modification found in diverse organisms ranging from bacteria and viruses to multicellular plants and humans. New developments in pseudouridine profiling provide quantitative tools to map mRNA pseudouridylation sites. Sparse biochemical studies establish the potential for mRNA pseudouridylation to affect most stages of the mRNA life cycle from birth to death. This recent progress sets the stage for deeper investigations into the molecular and cellular functions of specific mRNA pseudouridines, including in disease.

Cryo-EM structures of human DICER dicing a pre-miRNA substrate.

Dicer, a multi-domain ribonuclease III (RNase III) protein, is crucial for gene regulation via RNA interference. It processes hairpin-like precursors into microRNAs (miRNAs) and long double-stranded RNAs (dsRNAs) into small interfering RNAs (siRNAs). During the "dicing" process, the miRNA or siRNA substrate is stably anchored and cleaved by Dicer's RNase III domain. Although numerous studies have investigated long dsRNA cleavage by Dicer, the specific mechanism by which human Dicer (hDICER) processes pre-miRNA remains unelucidated. This review introduces the recently revealed hDICER structure bound to pre-miRNA uncovered through cryo-electron microscopy and compares it with previous reports describing Dicer. The domain-wise movements of the helicase and dsRNA-binding domain (dsRBD) and specific residues involved in substrate sequence recognition have been identified. During RNA substrate binding, the hDICER apical domains and dsRBD recognize the pre-miRNA termini and cleavage site, respectively. Residue rearrangements in positively charged pockets within the apical domain influence substrate recognition and cleavage site determination. The specific interactions between dsRBD positively charged residues and nucleotide bases near the cleavage site emphasize the significance of cis-acting elements in the hDICER processing mechanism. These findings provide valuable insights for understanding hDICER-related diseases.

Structural insights into intron catalysis and dynamics during splicing.

The group II intron ribonucleoprotein is an archetypal splicing system with numerous mechanistic parallels to the spliceosome, including excision of lariat introns1,2. Despite the importance of branching in RNA metabolism, structural understanding of this process has remained elusive. Here we present a comprehensive analysis of three single-particle cryogenic electron microscopy structures captured along the splicing pathway. They reveal the network of molecular interactions that specifies the branchpoint adenosine and positions key functional groups to catalyse lariat formation and coordinate exon ligation. The structures also reveal conformational rearrangements of the branch helix and the mechanism of splice site exchange that facilitate the transition from branching to ligation. These findings shed light on the evolution of splicing and highlight the conservation of structural components, catalytic mechanism and dynamical strategies retained through time in premessenger RNA splicing machines.

New insights into RNA processing by the eukaryotic tRNA splicing endonuclease.

Through its role in intron cleavage, tRNA splicing endonuclease (TSEN) plays a critical function in the maturation of intron-containing pre-tRNAs. The catalytic mechanism and core requirement for this process is conserved between archaea and eukaryotes, but for decades, it has been known that eukaryotic TSENs have evolved additional modes of RNA recognition, which have remained poorly understood. Recent research identified new roles for eukaryotic TSEN, including processing or degradation of additional RNA substrates, and determined the first structures of pre-tRNA-bound human TSEN complexes. These recent discoveries have changed our understanding of how the eukaryotic TSEN targets and recognizes substrates. Here, we review these recent discoveries, their implications, and the new questions raised by these findings.

Monovalent Ionic Atmosphere Modulates the Selection of Suboptimal RNA Sequences by Splicing Factors' RNA Recognition Motifs.

The U2AF2 splicing factor is involved in the RNA recognition of the pre-mRNA poly-pyrimidine signaling sequence. This protein contains two RRM domains connected by a flexible linker, which ensure the preferential selection of a poly-uridine sequence over a poly-cytosine one. In this work, all-atom simulations provide insights into the U2AF2 recognition mechanism and on the features underlying its selectivity. Our outcomes show that U2AF2's RNA recognition is driven by cooperative events modulated by RNA-protein and RNA-ion interactions. Stunningly, monovalent ions contribute to mediating the binding of the weakly binding polyC strand, thus contributing to the selection of suboptimal poly-pyrimidine tracts. This finding broadens our understanding of the diverse traits tuning splicing factors' selectivity and adaptability to precisely handle and process diverse pre-mRNA sequences.

Impact of the yeast S0/uS2-cluster ribosomal protein rpS21/eS21 on rRNA folding and the architecture of small ribosomal subunit precursors.

RpS0/uS2, rpS2/uS5, and rpS21/eS21 form a cluster of ribosomal proteins (S0-cluster) at the head-body junction near the central pseudoknot of eukaryotic small ribosomal subunits (SSU). Previous work in yeast indicated that S0-cluster assembly is required for the stabilisation and maturation of SSU precursors at specific post-nucleolar stages. Here, we analysed the role of S0-cluster formation for rRNA folding. Structures of SSU precursors isolated from yeast S0-cluster expression mutants or control strains were analysed by cryogenic electron microscopy. The obtained resolution was sufficient to detect individual 2'-O-methyl RNA modifications using an unbiased scoring approach. The data show how S0-cluster formation enables the initial recruitment of the pre-rRNA processing factor Nob1 in yeast. Furthermore, they reveal hierarchical effects on the pre-rRNA folding pathway, including the final maturation of the central pseudoknot. Based on these structural insights we discuss how formation of the S0-cluster determines at this early cytoplasmic assembly checkpoint if SSU precursors further mature or are degraded.

Sequence determinant of small RNA production by DICER.

RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs)1,2. However, our current knowledge of the specificity of Dicer is limited to the secondary structures of its substrates: a double-stranded RNA of approximately 22 base pairs with a 2-nucleotide 3' overhang and a terminal loop3-11. Here we found evidence pointing to an additional sequence-dependent determinant beyond these structural properties. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with pre-miRNA variants and human DICER (also known as DICER1). Our analyses revealed a deeply conserved cis-acting element, termed the 'GYM motif' (paired G, paired pyrimidine and mismatched C or A), near the cleavage site. The GYM motif promotes processing at a specific position and can override the previously identified 'ruler'-like counting mechanisms from the 5' and 3' ends of pre-miRNA3-6. Consistently, integrating this motif into short hairpin RNA or Dicer-substrate siRNA potentiates RNA interference. Furthermore, we find that the C-terminal double-stranded RNA-binding domain (dsRBD) of DICER recognizes the GYM motif. Alterations in the dsRBD reduce processing and change cleavage sites in a motif-dependent fashion, affecting the miRNA repertoire in cells. In particular, the cancer-associated R1855L substitution in the dsRBD strongly impairs GYM motif recognition. This study uncovers an ancient principle of substrate recognition by metazoan Dicer and implicates its potential in the design of RNA therapeutics.

Structure of the human DICER-pre-miRNA complex in a dicing state.

Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs)1,2. Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'3. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.

Structural studies of the spliceosome: Bridging the gaps.

The spliceosome is a multi-megadalton RNA-protein complex responsible for the removal of non-coding introns from pre-mRNAs. Due to its complexity and dynamic nature, it has proven to be a very challenging target for structural studies. Developments in single particle cryo-EM have overcome these previous limitations and paved the way towards a structural characterisation of the splicing machinery. Despite tremendous progress, many aspects of spliceosome structure and function remain elusive. In particular, the events leading to the definition of exon-intron boundaries, alternative and non-canonical splicing events, and cross-talk with other cellular machineries. Efforts are being made to address these knowledge gaps and further our mechanistic understanding of the spliceosome. Here, we summarise recent progress in the structural and functional analysis of the spliceosome.

Role of computational and structural biology in the development of small-molecule modulators of the spliceosome.

INTRODUCTION: RNA splicing is a pivotal step of eukaryotic gene expression during which the introns are excised from the precursor (pre-)RNA and the exons are joined together to form mature RNA products (i.e a protein-coding mRNA or long non-coding (lnc)RNAs). The spliceosome, a complex ribonucleoprotein machine, performs pre-RNA splicing with extreme precision. Deregulated splicing is linked to cancer, genetic, and neurodegenerative diseases. Hence, the discovery of small-molecules targeting core spliceosome components represents an appealing therapeutic opportunity. AREA COVERED: Several atomic-level structures of the spliceosome and distinct splicing-modulators bound to its protein/RNA components have been solved. Here, we review recent advances in the discovery of small-molecule splicing-modulators, discuss opportunities and challenges for their therapeutic applicability, and showcase how structural data and/or all-atom simulations can illuminate key facets of their mechanism, thus contributing to future drug-discovery campaigns. EXPERT OPINION: This review highlights the potential of modulating pre-RNA splicing with small-molecules, and anticipates how the synergy of computer and wet-lab experiments will enrich our understanding of splicing regulation/deregulation mechanisms. This information will aid future structure-based drug-discovery efforts aimed to expand the currently limited portfolio of selective splicing-modulators.

In vitro characterization of Dhr1 from Saccharomyces cerevisiae.

The RNA helicase Dhr1 from S. cerevisiae is an essential enzyme required for the assembly of the cytosolic small ribosomal subunit (SSU). A critical feature of the SSU is the central pseudoknot, an RNA fold that organizes the overall architecture of the subunit and connects all four domains of the 18S ribosomal RNA (rRNA). The initial folding of rRNA is guided, in part, by the U3 small nucleolar RNA, which base-pairs with the pre-rRNA in such a way as to preclude premature formation of the central pseudoknot. Thus, the essential role of Dhr1 is the unwinding of U3 from the pre-rRNA to allow folding of the central pseudoknot. Enzymes of the DEAH/RNA helicase A-like (RHA) family, to which Dhr1 belongs, are involved in splicing and ribosome biogenesis. They typically unwind RNA duplexes by translocation along a single strand of RNA in a 3' to 5' direction, driven by ATP hydrolysis. The substrate specificity of these enzymes requires tight regulation of their activity, by restricting access to their substrates, requiring adaptors to recruit them to their substrates and mechanisms of inhibiting and activating their activity. Purified Dhr1 is an active RNA-dependent ATPase with specific unwinding activity. Here, we provide detailed protocols for its purification and assays for its ATPase and unwinding activities.

Eukaryotic tRNA splicing - one goal, two strategies, many players.

Transfer RNAs (tRNAs) are transcribed as precursor molecules that undergo several maturation steps before becoming functional for protein synthesis. One such processing mechanism is the enzyme-catalysed splicing of intron-containing pre-tRNAs. Eukaryotic tRNA splicing is an essential process since intron-containing tRNAs cannot fulfil their canonical function at the ribosome. Splicing of pre-tRNAs occurs in two steps: The introns are first excised by a tRNA-splicing endonuclease and the exons are subsequently sealed by an RNA ligase. An intriguing complexity has emerged from newly identified tRNA splicing factors and their interplay with other RNA processing pathways during the past few years. This review summarises our current understanding of eukaryotic tRNA splicing and the underlying enzyme machinery. We highlight recent structural advances and how they have shaped our mechanistic understanding of tRNA splicing in eukaryotic cells. A special focus lies on biochemically distinct strategies for exon-exon ligation in fungi versus metazoans.

Atypical U3 snoRNA Suppresses the Process of Pterygium Through Modulating 18S Ribosomal RNA Synthesis.

Background: The progression and recurrence of pterygium mainly occur due to the abnormal proliferation and migration of stromal pterygium fibroblasts. This research explores the aberrant expression of small nucleolar RNA U3 (U3 snoRNA) in pterygium and elucidates the molecular mechanisms of U3 snoRNA in pterygium development. Methods: Primary human conjunctival fibroblasts (HCFs) and human pterygium fibroblasts (HPFs) were separated and cultured from fresh conjunctiva grafts and pterygium tissues. The PLKO.1 lentiviral system and CRISPR/Cas9 recombinant construct were, respectively, used to overexpress and silence U3 snoRNA in HPFs and HCFs for further specific phenotype analysis. RNA-seq and TMT-labeled quantitative protein mass spectrometry were utilized to evaluate the effect of U3 snoRNA on mRNA transcripts and protein synthesis. Results: Reduced U3 snoRNA in pterygium promotes HCF or HPF cells' proliferation, migration, and cell cycle but has no significant effect on apoptosis. U3 snoRNA modulates 18S rRNA synthesis through shearing precursor ribosomal RNA 47S rRNA at the 5' external transcribed spacer (5' ETS). Moreover, the altered U3 snoRNA causes mRNA and protein differential expression in HCF or HPF cells. Conclusions: The atypical U3 snoRNA regulates the translation of specific proteins to exert a suppressive function in pterygium through modulating the 18S rRNA synthesis. Here, we uncover a novel insight into U3 snoRNA biology in the development of pterygium.

miRNAFinder: A comprehensive web resource for plant Pre-microRNA classification.

microRNAs (miRNAs) are known as one of the small non-coding RNA molecules that control the expression of genes at the RNA level, while some operate at the DNA level. They typically range from 20 to 24 nucleotides in length and can be found in the plant and animal kingdoms as well as in some viruses. Computational approaches have overcome the limitations of the experimental methods and have performed well in identifying miRNAs. Compared to mature miRNAs, precursor miRNAs (pre-miRNAs) are long and have a hairpin loop structure with structural features. Therefore, most in-silico tools are implemented for pre-miRNA identification. This study presents a multilayer perceptron (MLP) based classifier implemented using 180 features under sequential, structural, and thermodynamic feature categories for plant pre-miRNA identification. This classifier has a 92% accuracy, a 94% specificity, and a 90% sensitivity. We have further tested this model with other small non-coding RNA types and obtained 78% accuracy. Furthermore, we introduce a novel dataset to train and test machine learning models, addressing the overlapping data issue in the positive training and testing datasets presented in PlantMiRNAPred for the classification of real and pseudo-plant pre-miRNAs. The new dataset and the classifier that can be used with any plant species are deployed on a web server freely accessible at http://mirnafinder.shyaman.me/.

Mutations in RNU7-1 Weaken Secondary RNA Structure, Induce MCP-1 and CXCL10 in CSF, and Result in Aicardi-Goutières Syndrome with Severe End-Organ Involvement.

BACKGROUND: Aicardi-Goutières syndrome (AGS) is a type I interferonopathy usually characterized by early-onset neurologic regression. Biallelic mutations in LSM11 and RNU7-1, components of the U7 small nuclear ribonucleoprotein (snRNP) complex, have been identified in a limited number of genetically unexplained AGS cases. Impairment of U7 snRNP function results in misprocessing of replication-dependent histone (RDH) pre-mRNA and disturbance of histone occupancy of nuclear DNA, ultimately driving cGAS-dependent type I interferon (IFN-I) release. OBJECTIVE: We performed a clinical, genetic, and immunological workup of 3 unrelated patients with uncharacterized AGS. METHODS: Whole exome sequencing (WES) and targeted Sanger sequencing of RNU7-1 were performed. Primary fibroblasts were used for mechanistic studies. IFN-I signature and STAT1/2 phosphorylation were assessed in peripheral blood. Cytokines were profiled on serum and cerebrospinal fluid (CSF). Histopathology was examined on brain and kidney tissue. RESULTS: Sequencing revealed compound heterozygous RNU7-1 mutations, resulting in impaired RDH pre-mRNA processing. The 3' stem-loop mutations reduced stability of the secondary U7 snRNA structure. A discrete IFN-I signature in peripheral blood was paralleled by MCP-1 (CCL2) and CXCL10 upregulation in CSF. Histopathological analysis of the kidney showed thrombotic microangiopathy. We observed dysregulated STAT phosphorylation upon cytokine stimulation. Clinical overview of all reported patients with RNU7-1-related disease revealed high mortality and high incidence of organ involvement compared to other AGS genotypes. CONCLUSIONS: Targeted RNU7-1 sequencing is recommended in genetically unexplained AGS cases. CSF cytokine profiling represents an additional diagnostic tool to identify aberrant IFN-I signaling. Clinical follow-up of RNU7-1-mutated patients should include screening for severe end-organ involvement including liver disease and nephropathy.