A Deep Neural Network for Predicting and Engineering Alternative Polyadenylation.

Alternative polyadenylation (APA) is a major driver of transcriptome diversity in human cells. Here, we use deep learning to predict APA from DNA sequence alone. We trained our model (APARENT, APA REgression NeT) on isoform expression data from over 3 million APA reporters. APARENT's predictions are highly accurate when tasked with inferring APA in synthetic and human 3'UTRs. Visualizing features learned across all network layers reveals that APARENT recognizes sequence motifs known to recruit APA regulators, discovers previously unknown sequence determinants of 3' end processing, and integrates these features into a comprehensive, interpretable, cis-regulatory code. We apply APARENT to forward engineer functional polyadenylation signals with precisely defined cleavage position and isoform usage and validate predictions experimentally. Finally, we use APARENT to quantify the impact of genetic variants on APA. Our approach detects pathogenic variants in a wide range of disease contexts, expanding our understanding of the genetic origins of disease.

Structural Basis of Transcription: RNA Polymerase Backtracking and Its Reactivation.

Regulatory sequences or erroneous incorporations during DNA transcription cause RNA polymerase backtracking and inactivation in all kingdoms of life. Reactivation requires RNA transcript cleavage. Essential transcription factors (GreA and GreB, or TFIIS) accelerate this reaction. We report four cryo-EM reconstructions of Escherichia coli RNA polymerase representing the entire reaction pathway: (1) a backtracked complex; a backtracked complex with GreB (2) before and (3) after RNA cleavage; and (4) a reactivated, substrate-bound complex with GreB before RNA extension. Compared with eukaryotes, the backtracked RNA adopts a different conformation. RNA polymerase conformational changes cause distinct GreB states: a fully engaged GreB before cleavage; a disengaged GreB after cleavage; and a dislodged, loosely bound GreB removed from the active site to allow RNA extension. These reconstructions provide insight into the catalytic mechanism and dynamics of RNA cleavage and extension and suggest how GreB targets backtracked complexes without interfering with canonical transcription.

Kinetic analysis of RNA cleavage by coronavirus Nsp15 endonuclease: Evidence for acid-base catalysis and substrate-dependent metal ion activation.

Understanding the functional properties of severe acute respiratory syndrome coronavirus 2 nonstructural proteins is essential for defining their roles in the viral life cycle, developing improved therapeutics and diagnostics, and countering future variants. Coronavirus nonstructural protein Nsp15 is a hexameric U-specific endonuclease whose functions, substrate specificity, mechanism, and dynamics are not fully defined. Previous studies report that Nsp15 requires Mn2+ ions for optimal activity; however, the effects of divalent ions on Nsp15 reaction kinetics have not been investigated in detail. Here, we analyzed the single- and multiple-turnover kinetics for model ssRNA substrates. Our data confirm that divalent ions are dispensable for catalysis and show that Mn2+ activates Nsp15 cleavage of two different ssRNA oligonucleotide substrates but not a dinucleotide. Biphasic kinetics of ssRNA substrates demonstrates that Mn2+ stabilizes alternative enzyme states that have faster substrate cleavage on the enzyme. However, we did not detect Mn2+-induced conformational changes using CD and fluorescence spectroscopy. The pH-rate profiles in the presence and absence of Mn2+ reveal active-site ionizable groups with similar pKas of ca. 4.8 to 5.2. An Rp stereoisomer phosphorothioate modification at the scissile phosphate had minimal effect on catalysis supporting a mechanism involving an anionic transition state. However, the Sp stereoisomer is inactive because of weak binding, consistent with models that position the nonbridging phosphoryl oxygen deep in the active site. Together, these data demonstrate that Nsp15 employs a conventional acid-base catalytic mechanism passing through an anionic transition state, and that divalent ion activation is substrate dependent.

TRANS-ACTING SIRNA3-derived short interfering RNAs confer cleavage of mRNAs in rice.

Plant TRANS-ACTING SIRNA3 (TAS3)-derived short interfering RNAs (siRNAs) include tasiR-AUXIN RESPONSE FACTORs (ARFs), which are functionally conserved in targeting ARF genes, and a set of non-tasiR-ARF siRNAs, which have rarely been studied. In this study, TAS3 siRNAs were systematically characterized in rice (Oryza sativa). Small RNA sequencing results showed that an overwhelming majority of TAS3 siRNAs belong to the non-tasiR-ARF group, while tasiR-ARFs occupy a diminutive fraction. Phylogenetic analysis of TAS3 genes across dicot and monocot plants revealed that the siRNA-generating regions were highly conserved in grass species, especially in the Oryzoideae. Target genes were identified for not only tasiR-ARFs but also non-tasiR-ARF siRNAs by analyzing rice Parallel Analysis of RNA Ends datasets, and some of these siRNA-target interactions were experimentally confirmed using tas3 mutants generated by genome editing. Consistent with the de-repression of target genes, phenotypic alterations were observed for mutants in three TAS3 loci in comparison to wild-type rice. The regulatory role of ribosomes in the TAS3 siRNA-target interactions was further revealed by the fact that TAS3 siRNA-mediated target cleavage, in particular tasiR-ARFs targeting ARF2/3/14/15, occurred extensively in rice polysome samples. Altogether, our study sheds light into TAS3 genes in plants and expands our knowledge about rice TAS3 siRNA-target interactions.

Co-transcriptional RNA cleavage by Drosha homolog Pac1 triggers transcription termination in fission yeast.

Transcription termination of protein-coding genes in eukaryotic cells usually relies on a tight coordination between the cleavage and polyadenylation of the pre-mRNA, and 5'-3' degradation of the downstream nascent transcript. Here we investigated the contribution of the essential fission yeast endonuclease Pac1, a homolog of human Drosha that cleaves hairpin RNA structures, in triggering polyadenylation-independent transcription termination. Using ChIP-sequencing in Pac1-deficient cells, we found that Pac1 triggers transcription termination at snRNA and snoRNA genes as well as at specific protein-coding genes. Notably, we found that Pac1-dependent premature termination occurred at two genes encoding conserved transmembrane transporters whose expression were strongly repressed by Pac1. Analysis by genome editing indicated that a stem-loop structure in the nascent transcript directs Pac1-mediated cleavage and that the regions upstream and downstream of the Pac1 cleavage site in the targeted mRNAs were stabilized by mutation of nuclear 3'-5' and 5'-3' exonucleases, respectively. Our findings unveil a premature transcription termination pathway that uncouples co-transcriptional RNA cleavage from polyadenylation, triggering rapid nuclear RNA degradation.

Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA in Aedes aegypti mosquitoes.

In the germline of animals, PIWI interacting (pi)RNAs protect the genome against the detrimental effects of transposon mobilization. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the production of responder piRNAs via ping-pong amplification. Responder piRNA 3' end formation by the nuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of transposon piRNA sequences. In Aedes aegypti mosquitoes, piRNAs are generated from viral RNA, yet, it is unknown how viral piRNA 3' ends are formed and whether viral RNA cleavage gives rise to trailer piRNA production. Here we report that in Ae. aegypti, virus- and transposon-derived piRNAs have sharp 3' ends, and are biased for downstream uridine residues, features reminiscent of Zucchini cleavage of precursor piRNAs in Drosophila. We designed a reporter system to study viral piRNA 3' end formation and found that targeting viral RNA by abundant endogenous piRNAs triggers the production of responder and trailer piRNAs. Using this reporter, we identified the Ae. aegypti orthologs of Zucchini and Nibbler, two nucleases involved in piRNA 3' end formation. Our results furthermore suggest that autonomous piRNA production from viral RNA can be triggered and expanded by an initial cleavage event guided by genome-encoded piRNAs.

Cutting in-line with iron: ribosomal function and non-oxidative RNA cleavage.

Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg2+ as a primary mediator of ribosomal structure and function. Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosome's early evolution in a high Fe2+ environment, and the continued use of Fe2+ by obligate anaerobes inhabiting high Fe2+ niches. Here, we show that (i) Fe2+ cleaves RNA by in-line cleavage, a non-oxidative mechanism that has not previously been shown experimentally for this metal, (ii) the first-order in-line rate constant with respect to divalent cations is >200 times greater with Fe2+ than with Mg2+, (iii) functional ribosomes are associated with Fe2+ after purification from cells grown under low O2 and high Fe2+ and (iv) a small fraction of Fe2+ that is associated with the ribosome is not exchangeable with surrounding divalent cations, presumably because those ions are tightly coordinated by rRNA and deeply buried in the ribosome. In total, these results expand the ancient role of iron in biochemistry and highlight a possible new mechanism of iron toxicity.

Genome-wide identification of short 2',3'-cyclic phosphate-containing RNAs and their regulation in aging.

RNA molecules generated by ribonuclease cleavage sometimes harbor a 2',3'-cyclic phosphate (cP) at their 3'-ends. Those cP-containing RNAs (cP-RNAs) form a hidden layer of transcriptome because standard RNA-seq cannot capture them as a result of cP's prevention of an adapter ligation reaction. Here we provide genome-wide analyses of short cP-RNA transcriptome across multiple mouse tissues. Using cP-RNA-seq that can exclusively sequence cP-RNAs, we identified numerous novel cP-RNA species which are mainly derived from cytoplasmic tRNAs, mRNAs, and rRNAs. Determination of the processing sites of substrate RNAs for cP-RNA generation revealed highly-specific RNA cleavage events between cytidine and adenosine in cP-RNA biogenesis. cP-RNAs were not evenly derived from the overall region of substrate RNAs but rather from specific sites, implying that cP-RNAs are not from random degradation but are produced through a regulated biogenesis pathway. The identified cP-RNAs were abundantly accumulated in mouse tissues, and the expression levels of cP-RNAs showed age-dependent reduction. These analyses of cP-RNA transcriptome unravel a novel, abundant class of non-coding RNAs whose expression could have physiological roles.

Downstream sequence-dependent RNA cleavage and pausing by RNA polymerase I.

The sequence of the DNA template has long been thought to influence the rate of transcription by DNA-dependent RNA polymerases, but the influence of DNA sequence on transcription elongation properties of eukaryotic RNA polymerase I (Pol I) from Saccharomyces cerevisiae has not been defined. In this study, we observe changes in dinucleotide production, transcription elongation complex stability, and Pol I pausing in vitro in response to downstream DNA. In vitro studies demonstrate that AT-rich downstream DNA enhances pausing by Pol I and inhibits Pol I nucleolytic cleavage activity. Analysis of Pol I native elongating transcript sequencing data in Saccharomyces cerevisiae suggests that these downstream sequence elements influence Pol I in vivo Native elongating transcript sequencing studies reveal that Pol I occupancy increases as downstream AT content increases and decreases as downstream GC content increases. Collectively, these data demonstrate that the downstream DNA sequence directly impacts the kinetics of transcription elongation prior to the sequence entering the active site of Pol I both in vivo and in vitro.

A cell-based splicing reporter system to identify regulators of cis-splicing between adjacent genes.

Chimeric RNAs generated by cis-splicing between adjacent genes (cis-SAGe) are increasingly recognized as a widespread phenomenon. These chimeric messenger RNAs are present in normal human cells, and are also detected in various cancers. The mechanisms for how this group of chimeras is formed are not yet clear, in part due to the lack of a tractable system for their experimental investigation. Here we developed a fast, easy and versatile cell-based reporter system to identify regulators of cis-SAGe. The reporter, consisting of four main cassettes, simultaneously measures the effects of a candidate regulator on cis-SAGe and canonical splicing. Using this cell-based assay, we screened 102 candidate factors involved in RNA pol II cleavage and termination, elongation, splicing, alternative splicing and R-loop formation. We discovered that two factors, SRRM1 and SF3B1, affect not only cis-SAGe chimeras, but also other types of chimeric RNAs in a genome-wide fashion. This system can be used for studying trans-acting factors and cis-acting sequence elements and factors, as well as for screening small molecule inhibitors.

Dynamical ensemble of the active state and transition state mimic for the RNA-cleaving 8-17 DNAzyme in solution.

We perform molecular dynamics simulations, based on recent crystallographic data, on the 8-17 DNAzyme at four states along the reaction pathway to determine the dynamical ensemble for the active state and transition state mimic in solution. A striking finding is the diverse roles played by Na+ and Pb2+ ions in the electrostatically strained active site that impact all four fundamental catalytic strategies, and share commonality with some features recently inferred for naturally occurring hammerhead and pistol ribozymes. The active site Pb2+ ion helps to stabilize in-line nucleophilic attack, provides direct electrostatic transition state stabilization, and facilitates leaving group departure. A conserved guanine residue is positioned to act as the general base, and is assisted by a bridging Na+ ion that tunes the pKa and facilitates in-line fitness. The present work provides insight into how DNA molecules are able to solve the RNA-cleavage problem, and establishes functional relationships between the mechanism of these engineered DNA enzymes with their naturally evolved RNA counterparts. This adds valuable information to our growing body of knowledge on general mechanisms of phosphoryl transfer reactions catalyzed by RNA, proteins and DNA.

RNA polymerase III subunits C37/53 modulate rU:dA hybrid 3' end dynamics during transcription termination.

RNA polymerase (RNAP) III synthesizes tRNAs and other transcripts, and mutations to its subunits cause human disorders. The RNAP III subunit-heterodimer C37/53 functions in initiation, elongation and in termination-associated reinitiation with subunit C11. C37/53 is related to heterodimers associated with RNAPs I and II, and C11 is related to TFIIS and Rpa12.2, the active site RNA 3' cleavage factors for RNAPs II and I. Critical to termination is stability of the RNA:DNA hybrid bound in the active center, which is loose for RNAP III relative to other RNAPs. Here, we examined RNAP III lacking C37/53/C11 and various reconstituted forms during termination. First, we established a minimal terminator as 5T and 3A on the non-template and template DNA strands, respectively. We demonstrate that C11 stimulates termination, and does so independently of its RNA cleavage activity. We found that C37/53 sensitizes RNAP III termination to RNA:DNA hybrid strength and promotes RNA 3' end pairing/annealing with the template. The latter counteracts C11-insensitive arrest in the proximal part of the oligo(T)-tract, promoting oligo(rU:dA) extension toward greater hybrid instability and RNA release. The data also indicate that RNA 3' end engagement with the active site is a determinant of termination. Broader implications are also discussed.

Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence.

The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.

m5U54 tRNA Hypomodification by Lack of TRMT2A Drives the Generation of tRNA-Derived Small RNAs.

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification and tsRNA formation. More specifically, m5U54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5'tRNA-derived stress-induced RNAs (5'tiRNAs), namely 5'tiRNA-GlyGCC and 5'tiRNA-GluCTC, among others. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.

The structural basis of CstF-77 modulation of cleavage and polyadenylation through stimulation of CstF-64 activity.

Cleavage and polyadenylation (C/P) of mRNA is an important cellular process that promotes increased diversity of mRNA isoforms and could change their stability in different cell types. The cleavage stimulation factor (CstF) complex, part of the C/P machinery, binds to U- and GU-rich sequences located downstream from the cleavage site through its RNA-binding subunit, CstF-64. Less is known about the function of the other two subunits of CstF, CstF-77 and CstF-50. Here, we show that the carboxy-terminus of CstF-77 plays a previously unrecognized role in enhancing C/P by altering how the RNA recognition motif (RRM) of CstF-64 binds RNA. In support of this finding, we also show that CstF-64 relies on CstF-77 to be transported to the nucleus; excess CstF-64 localizes to the cytoplasm, possibly via interaction with cytoplasmic RNAs. Reverse genetics and nuclear magnetic resonance studies of recombinant CstF-64 (RRM-Hinge) and CstF-77 (monkeytail-carboxy-terminal domain) indicate that the last 30 amino acids of CstF-77 increases the stability of the RRM, thus altering the affinity of the complex for RNA. These results provide new insights into the mechanism by which CstF regulates the location of the RNA cleavage site during C/P.

Efficient gene correction of an aberrant splice site in ß-thalassaemia iPSCs by CRISPR/Cas9 and single-strand oligodeoxynucleotides.

ß-thalassaemia is a prevalent hereditary haematological disease caused by mutations in the human haemoglobin ß (HBB) gene. Among them, the HBB IVS2-654 (C > T) mutation, which is in the intron, creates an aberrant splicing site. Bone marrow transplantation for curing ß-thalassaemia is limited due to the lack of matched donors. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as a widely used tool for gene editing, is able to target specific sequence and create double-strand break (DSB), which can be combined with the single-stranded oligodeoxynucleotide (ssODN) to correct mutations. In this study, according to two different strategies, the HBB IVS2-654 mutation was seamlessly corrected in iPSCs by CRISPR/Cas9 system and ssODN. To reduce the occurrence of secondary cleavage, a more efficient strategy was adopted. The corrected iPSCs kept pluripotency and genome stability. Moreover, they could differentiate normally. Through CRISPR/Cas9 system and ssODN, our study provides improved strategies for gene correction of ß-Thalassaemia, and the expression of the HBB gene can be restored, which can be used for gene therapy in the future.

JMJD5 cleaves monomethylated histone H3 N-tail under DNA damaging stress.

The histone H3 N-terminal protein domain (N-tail) is regulated by multiple posttranslational modifications, including methylation, acetylation, phosphorylation, and by proteolytic cleavage. However, the mechanism underlying H3 N-tail proteolytic cleavage is largely elusive. Here, we report that JMJD5, a Jumonji C (JmjC) domain-containing protein, is a Cathepsin L-type protease that mediates histone H3 N-tail proteolytic cleavage under stress conditions that cause a DNA damage response. JMJD5 clips the H3 N-tail at the carboxyl side of monomethyl-lysine (Kme1) residues. In vitro H3 peptide digestion reveals that JMJD5 exclusively cleaves Kme1 H3 peptides, while little or no cleavage effect of JMJD5 on dimethyl-lysine (Kme2), trimethyl-lysine (Kme3), or unmethyl-lysine (Kme0) H3 peptides is observed. Although H3 Kme1 peptides of K4, K9, K27, and K36 can all be cleaved by JMJD5 in vitro, K9 of H3 is the major cleavage site in vivo, and H3.3 is the major H3 target of JMJD5 cleavage. Cleavage is enhanced at gene promoters bound and repressed by JMJD5 suggesting a role for H3 N-tail cleavage in gene expression regulation.

Cleavage and polyadenylation: Ending the message expands gene regulation.

Cleavage and polyadenylation (pA) is a fundamental step that is required for the maturation of primary protein encoding transcripts into functional mRNAs that can be exported from the nucleus and translated in the cytoplasm. 3'end processing is dependent on the assembly of a multiprotein processing complex on the pA signals that reside in the pre-mRNAs. Most eukaryotic genes have multiple pA signals, resulting in alternative cleavage and polyadenylation (APA), a widespread phenomenon that is important to establish cell state and cell type specific transcriptomes. Here, we review how pA sites are recognized and comprehensively summarize how APA is regulated and creates mRNA isoform profiles that are characteristic for cell types, tissues, cellular states and disease.

In Vitro Selection of a DNAzyme Cooperatively Binding Two Lanthanide Ions for RNA Cleavage.

Trivalent lanthanide ions (Ln(3+)) were recently employed to select RNA-cleaving DNAzymes, and three new DNAzymes have been reported so far. In this work, dysprosium (Dy(3+)) was used with a library containing 50 random nucleotides. After six rounds of in vitro selection, a new DNAzyme named Dy10a was obtained and characterized. Dy10a has a bulged hairpin structure cleaving a RNA/DNA chimeric substrate. Dy10a is highly active in the presence of the five Ln(3+) ions in the middle of the lanthanide series (Sm(3+), Eu(3+), Gd(3+), Tb(3+), and Dy(3+)), while its activity descends on the two sides. The cleavage rate reaches 0.6 min(-1) at pH 6 with just 200 nM Sm(3+), which is the fastest among all known Ln(3+)-dependent enzymes. Dy10a binds two Ln(3+) ions cooperatively. When a phosphorothioate (PS) modification is introduced at the cleavage junction, the activity decreases by >2500-fold for both the Rp and Sp diastereomers, and thiophilic Cd(2+) cannot rescue the activity. The pH-rate profile has a slope of 0.37 between pH 4.2 and 5.2, and the slope was even lower at higher pH. On the basis of these data, a model of metal binding is proposed. Finally, a catalytic beacon sensor that can detect Ho(3+) down to 1.7 nM is constructed.

Generation of catalytic human Ago4 identifies structural elements important for RNA cleavage.

Argonaute proteins bind small RNAs and mediate cleavage of complementary target RNAs. The human Argonaute protein Ago4 is catalytically inactive, although it is highly similar to catalytic Ago2. Here, we have generated Ago2-Ago4 chimeras and analyzed their cleavage activity in vitro. We identify several specific features that inactivate Ago4: the catalytic center, short sequence elements in the N-terminal domain, and an Ago4-specific insertion in the catalytic domain. In addition, we show that Ago2-mediated cleavage of the noncanonical miR-451 precursor can be carried out by any catalytic human Ago protein. Finally, phylogenetic analyses establish evolutionary distances between the Ago proteins. Interestingly, these distances do not fully correlate with the structural changes inactivating them, suggesting functional adaptations of individual human Ago proteins.