End Maturation Research Articles

Modulation of alternative cleavage and polyadenylation events by dCas9-mediated CRISPRpas.

The CRISPR/Cas9 technology is revolutionizing genomic engineering. The high efficiency and selectivity of the system have inspired the development of various derived tools for gene regulation at different levels, such as transcriptional activation or inhibition, epigenetic modification, splicing, and base editing. Cleavage and polyadenylation (CPA) is an essential 3' end maturation step for almost all eukaryotic mRNAs. CPA is tightly coupled with transcriptional termination, and its activity impacts gene expression. Over half of all human genes display alternative polyadenylation (APA), where multiple cleavage and polyadenylation sites (PASs) lead to mRNA isoforms with variable termini. APA isoforms often have distinct metabolisms, and their relative abundance can change drastically in different cells. Here, we describe a method based on delivering a catalytically dead Cas9 (dCas9) to genomic regions nears the PAS, which alters APA site usage in 3'UTRs or introns. This method, named CRISPRpas, allows investigators to examine functional significance of APA isoforms of individual genes. We also describe using the bioinformatics program APAlyzer to examine APA events of interest with RNA-seq data.

ISG20 and nuclear exosome promote destabilization of nascent transcripts for spliceosomal U snRNAs and U1 variants

Primary RNA transcripts are processed in a plethora of ways to become mature functional forms. In one example, human spliceosomal U snRNAs are matured at their 3'-end by an exonuclease termed TOE1. This process is important because mutations in TOE1 gene can cause a human genetic disease, pontocerebellar hypoplasia (PCH). Nevertheless, TOE1 may not be the only maturation exonuclease for U snRNAs in the cell. Here, we biochemically identify two exonucleolytic factors, Interferon-stimulated gene 20-kDa protein (ISG20) and the nuclear exosome as such candidates, using a newly developed in vitro system that recapitulates 3'-end maturation of U1 snRNA. However, extensive 3'-end sequencing of endogenous U1 snRNA of the knockdown (KD) cells revealed that these factors are not the maturation factors per se. Instead, the nascent transcripts of the spliceosomal U snRNAs as well as of unstable U1 variants were found to increase in quantity upon KD of the factors. These results indicated that ISG20 and the nuclear exosome promote the degradation of nascent spliceosomal U snRNAs and U1 variants, and therefore implied their role in the quality control of newly synthesized U snRNAs.

Generation of a Single-Cell RNAseq Atlas of Murine Salivary Gland Development.

SummaryUnderstanding the dynamic transcriptional landscape throughout organ development will provide a template for regenerative therapies. Here, we generated a single-cell RNA sequencing atlas of murine submandibular glands identifying transcriptional profiles that revealed cellular heterogeneity during landmark developmental events: end bud formation, branching morphogenesis, cytodifferentiation, maturation, and homeostasis. Trajectory inference analysis suggests plasticity among acinar and duct populations. We identify transcription factors correlated with acinar differentiation including Spdef, Etv1, and Xbp1, and loss of Ybx1, Eno1, Sox11, and Atf4. Furthermore, we characterize two intercalated duct populations defined by either Gfra3 and Kit, or Gstt1. This atlas can be used to investigate specific cell functions and comparative studies predicting common mechanisms involved in development of branching organs.

TREND-DB-a transcriptome-wide atlas of the dynamic landscape of alternative polyadenylation.

Alternative polyadenylation (APA) profoundly expands the transcriptome complexity. Perturbations of APA can disrupt biological processes, ultimately resulting in devastating disorders. A major challenge in identifying mechanisms and consequences of APA (and its perturbations) lies in the complexity of RNA 3′ end processing, involving poorly conserved RNA motifs and multi-component complexes consisting of far more than 50 proteins. This is further complicated in that RNA 3′ end maturation is closely linked to transcription, RNA processing and even epigenetic (histone/DNA/RNA) modifications. Here, we present TREND-DB (http://shiny.imbei.uni-mainz.de:3838/trend-db), a resource cataloging the dynamic landscape of APA after depletion of >170 proteins involved in various facets of transcriptional, co- and post-transcriptional gene regulation, epigenetic modifications and further processes. TREND-DB visualizes the dynamics of transcriptome 3′ end diversification (TREND) in a highly interactive manner; it provides a global APA network map and allows interrogating genes affected by specific APA-regulators and vice versa. It also permits condition-specific functional enrichment analyses of APA-affected genes, which suggest wide biological and clinical relevance across all RNAi conditions. The implementation of the UCSC Genome Browser provides additional customizable layers of gene regulation accounting for individual transcript isoforms (e.g. epigenetics, miRNA-binding sites and RNA-binding proteins). TREND-DB thereby fosters disentangling the role of APA for various biological programs, including potential disease mechanisms, and helps identify their diagnostic and therapeutic potential.

Setaria viridis chlorotic and seedling-lethal mutants define critical functions for chloroplast gene expression.

Deep insights into chloroplast biogenesis have been obtained by mutant analysis; however, in C4 plants a relevant mutant collection has only been developed and exploited for maize. Here, we report the initial characterization of an ethyl methyl sulfonate-induced mutant population for the C4 model Setaria viridis. Approximately 1000 M2 families were screened for the segregation of pale-green seedlings in the M3 generation, and a subset of these was identified to be deficient in post-transcriptional steps of chloroplast gene expression. Causative mutations were identified for three lines using deep sequencing-based bulked segregant analysis, and in one case confirmed by transgenic complementation. Using chloroplast RNA-sequencing and other molecular assays, we describe phenotypes of mutants deficient in PSRP7, a plastid-specific ribosomal protein, OTP86, an RNA editing factor, and cpPNP, the chloroplast isozyme of polynucleotide phosphorylase. The psrp mutant is globally defective in chloroplast translation, and has varying deficiencies in the accumulation of chloroplast-encoded proteins. The otp86 mutant, like its Arabidopsis counterpart, is specifically defective in editing of the rps14 mRNA; however, the conditional pale-green mutant phenotype contrasts with the normal growth of the Arabidopsis mutant. The pnp mutant exhibited multiple defects in 3' end maturation as well as other qualitative changes in the chloroplast RNA population. Overall, our collection opens the door to global analysis of photosynthesis and early seedling development in an emerging C4 model.

Characterization of the Methanomicrobial Archaeal RNase Zs for Processing the CCA-Containing tRNA Precursors.

RNase Z is a widely distributed and usually essential endoribonuclease involved in the 3′-end maturation of transfer RNAs (tRNAs). A CCA triplet that is needed for tRNA aminoacylation in protein translation is added by a nucleotidyl-transferase after the 3′-end processing by RNase Z. However, a considerable proportion of the archaeal pre-tRNAs genetically encode a CCA motif, while the enzymatic characteristics of the archaeal RNase (aRNase) Zs in processing CCA-containing pre-tRNAs remain unclear. This study intensively characterized two methanomicrobial aRNase Zs, the Methanolobus psychrophilus mpy-RNase Z and the Methanococcus maripaludis mmp-RNase Z, particularly focusing on the properties of processing the CCA-containing pre-tRNAs, and in parallel comparison with a bacterial bsu-RNase Z from Bacillus subtilis. Kinetic analysis found that Co2+ supplementation enhanced the cleavage efficiency of mpy-RNase Z, mmp-RNase Z, and bsu-RNase Z for 1400-, 2990-, and 34-fold, respectively, and Co2+ is even more indispensable to the aRNase Zs than to bsu-RNase Z. Mg2+ also elevated the initial cleavage velocity (V0) of bsu-RNase Z for 60.5-fold. The two aRNase Zs exhibited indiscriminate efficiencies in processing CCA-containing vs. CCA-less pre-tRNAs. However, V0 of bsu-RNase Z was markedly reduced for 1520-fold by the CCA motif present in pre-tRNAs under Mg2+ supplementation, but only 5.8-fold reduced under Co2+ supplementation, suggesting Co2+ could ameliorate the CCA motif inhibition on bsu-RNase Z. By 3′-RACE, we determined that the aRNase Zs cleaved just downstream the discriminator nucleotide and the CCA triplet in CCA-less and CCA-containing pre-tRNAs, thus exposing the 3′-end for linking CCA and the genetically encoded CCA triplet, respectively. The aRNase Zs, but not bsu-RNase Z, were also able to process the intron-embedded archaeal pre-tRNAs, and even process pre-tRNAs that lack the D, T, or anticodon arm, but strictly required the acceptor stem. In summary, the two methanomicrobial aRNase Zs use cobalt as a metal ligand and process a broad spectrum of pre-tRNAs, and the characteristics would extend our understandings on aRNase Zs.

Alternative splicing of DSP1 enhances snRNA accumulation by promoting transcription termination and recycle of the processing complex

Small nuclear RNAs (snRNAs) are the basal components of the spliceosome and play crucial roles in splicing. Their biogenesis is spatiotemporally regulated. However, related mechanisms are still poorly understood. Defective in snRNA processing (DSP1) is an essential component of the DSP1 complex that catalyzes plant snRNA 3'-end maturation by cotranscriptional endonucleolytic cleavage of the primary snRNA transcripts (presnRNAs). Here, we show that DSP1 is subjected to alternative splicing in pollens and embryos, resulting in two splicing variants, DSP1α and DSP1β. Unlike DSP1α, DSP1β is not required for presnRNA 3'-end cleavage. Rather, it competes with DSP1α for the interaction with CPSF73-I, the catalytic subunit of the DSP1 complex, which promotes efficient release of CPSF73-I and the DNA-dependent RNA polymerease II (Pol II) from the 3' end of snRNA loci thereby facilitates snRNA transcription termination, resulting in increased snRNA levels in pollens. Taken together, this study uncovers a mechanism that spatially regulates snRNA accumulation.

RNase AM, a 5' to 3' exonuclease, matures the 5' end of all three ribosomal RNAs in E. coli.

Bacterial ribosomal RNAs (rRNAs) are transcribed as precursors and require processing by Ribonucleases (RNases) to generate mature and functional rRNAs. Although the initial steps of rRNA processing in Escherichia coli (E. coli) were described several decades ago, the enzymes responsible for the final steps of 5S and 23S rRNA 5′-end maturation have remained unknown. Here, I show that RNase AM, a recently identified 5′ to 3′ exonuclease, performs the last step of 5S rRNA 5′-end maturation. RNase AM was also found to generate the mature 5′ end of 23S rRNA, subsequent to a newly identified prior processing step. Additionally, RNase AM was found to mature the 5′ end of 16S rRNA, a reaction previously attributed to RNase G. These findings indicate a major role for RNase AM in cellular RNA metabolism and establish a biological role for the first 5′ to 3′ RNA exonuclease identified in E. coli.

Small-Molecule PAPD5 Inhibitors Restore Telomerase Activity in Patient Stem Cells.

Genetic lesions that reduce telomerase activity inhibit stem cell replication and cause a range of incurable diseases, including dyskeratosis congenita (DC) and pulmonary fibrosis (PF). Modalities to restore telomerase in stem cells throughout the body remain unclear. Here, we describe small-molecule PAPD5 inhibitors that demonstrate telomere restoration invitro, in stem cell models, and invivo. PAPD5 is a non-canonical polymerase that oligoadenylates and destabilizes telomerase RNA component (TERC). We identified BCH001, a specific PAPD5 inhibitor that restored telomerase activity and telomere length in DC patient induced pluripotent stem cells. When human blood stem cells engineered to carry DC-causing PARN mutations were xenotransplanted into immunodeficient mice, oral treatment with a repurposed PAPD5 inhibitor, the dihydroquinolizinone RG7834, rescued TERC 3' end maturation and telomere length. These findings pave the way for developing systemic telomere therapeutics to counteract stem cell exhaustion in DC, PF, and possibly other aging-related diseases.

Generation of pre-tRNAs from polycistronic operons is the essential function of RNase P in Escherichia coli.

Ribonuclease P (RNase P) is essential for the 5′-end maturation of tRNAs in all kingdoms of life. In Escherichia coli, temperature sensitive mutations in either its protein (rnpA49) and or RNA (rnpB709) subunits lead to inviability at nonpermissive temperatures. Using the rnpA49 temperature sensitive allele, which encodes a partially defective RNase P at the permissive temperature, we show here for the first time that the processing of RNase P-dependent polycistronic tRNA operons to release pre-tRNAs is the essential function of the enzyme, since the majority of 5′-immature tRNAs can be aminoacylated unless their 5′-extensions ≥8 nt. Surprisingly, the failure of 5′-end maturation elicits increased polyadenylation of some pre-tRNAs by poly(A) polymerase I (PAP I), which exacerbates inviability. The absence of PAP I led to improved aminoacylation of 5′-immature tRNAs. Our data suggest a more dynamic role for PAP I in maintaining functional tRNA levels in the cell.

A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana.

Potato spindle tuber viroid (PSTVd) is a circular non-coding RNA of 359 nucleotides that replicates and spreads systemically in host plants, thus all functions required to establish an infection are mediated by sequence and structural elements in the genome. The PSTVd secondary structure contains 26 Watson-Crick base-paired stems and 27 loops. Most of the loops are believed to form three-dimensional (3D) structural motifs through non-Watson-Crick base pairing, base stacking, and other local interactions. Homology-based prediction using the JAR3D online program revealed that loop 27 (nucleotides 177–182) most likely forms a 3D structure similar to the loop of a conserved hairpin located in the 3' untranslated region of histone mRNAs in animal cells. This stem-loop, which is involved in 3'-end maturation, is not found in polyadenylated plant histone mRNAs. Mutagenesis showed that PSTVd genomes containing base substitutions in loop 27 predicted by JAR3D to disrupt the 3D structure were unable to replicate in Nicotiana benthamiana leaves following mechanical rub inoculation, with one exception: a U178G/U179G double mutant was replication-competent and able to spread within the upper epidermis of inoculated leaves, but was confined to this cell layer. Remarkably, direct delivery of the U178G/U179G mutant into the vascular system by needle puncture inoculation allowed it to spread systemically and enter mesophyll cells and epidermal cells of upper leaves. These findings highlight the importance of RNA 3D structure for PSTVd replication and intercellular trafficking and indicate that loop 27 is required for epidermal exit, but not epidermal entry or transit between other cell types. Thus, requirements for RNA trafficking between epidermal and underlying palisade mesophyll cells are unique and directional. Our findings further suggest that 3D structure and RNA-protein interactions constrain RNA sequence evolution, and validate JAR3D as a tool to predict RNA 3D structure.

ZCCHC8, the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

Short telomere syndromes manifest as familial idiopathic pulmonary fibrosis; they are the most common premature aging disorders. We used genome-wide linkage to identify heterozygous loss of function of ZCCHC8, a zinc-knuckle containing protein, as a cause of autosomal dominant pulmonary fibrosis. ZCCHC8 associated with TR and was required for telomerase function. In ZCCHC8 knockout cells and in mutation carriers, genomically extended telomerase RNA (TR) accumulated at the expense of mature TR, consistent with a role for ZCCHC8 in mediating TR 3' end targeting to the nuclear RNA exosome. We generated Zcchc8-null mice and found that heterozygotes, similar to human mutation carriers, had TR insufficiency but an otherwise preserved transcriptome. In contrast, Zcchc8-/- mice developed progressive and fatal neurodevelopmental pathology with features of a ciliopathy. The Zcchc8-/- brain transcriptome was highly dysregulated, showing accumulation and 3' end misprocessing of other low-abundance RNAs, including those encoding cilia components as well as the intronless replication-dependent histones. Our data identify a novel cause of human short telomere syndromes-familial pulmonary fibrosis and uncover nuclear exosome targeting as an essential 3' end maturation mechanism that vertebrate TR shares with replication-dependent histones.

Interplay between substrate recognition, 5' end tRNA processing and methylation activity of human mitochondrial RNase P.

Human mitochondrial ribonuclease P (mtRNase P) is an essential three-protein complex that catalyzes the 5′ end maturation of mitochondrial precursor tRNAs (pre-tRNAs). Mitochondrial RNase P Protein 3 (MRPP3), a protein-only RNase P (PRORP), is the nuclease component of the mtRNase P complex and requires a two-protein S-adenosyl-methionine (SAM)-dependent methyltransferase MRPP1/2 subcomplex to function. Dysfunction of mtRNase P is linked to several human mitochondrial diseases, such as mitochondrial myopathies. Despite its central role in mitochondrial RNA processing, little is known about how the protein subunits of mtRNase P function synergistically. Here, we use purified mtRNase P to demonstrate that mtRNase P recognizes, cleaves, and methylates some, but not all, mitochondrial pre-tRNAs in vitro. Additionally, mtRNase P does not process all mitochondrial pre-tRNAs uniformly, suggesting the possibility that some pre-tRNAs require additional factors to be cleaved in vivo. Consistent with this, we found that addition of the TRMT10C (MRPP1) cofactor SAM enhances the ability of mtRNase P to bind and cleave some mitochondrial pre-tRNAs. Furthermore, the presence of MRPP3 can enhance the methylation activity of MRPP1/2. Taken together, our data demonstrate that the subunits of mtRNase P work together to efficiently recognize, process, and methylate human mitochondrial pre-tRNAs.

Separating the Wheat from the Chaff: RNA Editing and Selection of Translatable mRNA in Trypanosome Mitochondria.

In the mitochondria of trypanosomes and related kinetoplastid protists, most mRNAs undergo a long and sophisticated maturation pathway before they can be productively translated by mitochondrial ribosomes. Some of the aspects of this pathway (identity of the promotors, transcription initiation, and termination signals) remain obscure, and some (post-transcriptional modification by U-insertion/deletion, RNA editing, 3′-end maturation) have been illuminated by research during the last decades. The RNA editing creates an open reading frame for a productive translation, but the fully edited mRNA often represents a minor fraction in the pool of pre-edited and partially edited precursors. Therefore, it has been expected that the final stages of the mRNA processing generate molecular hallmarks, which allow for the efficient and selective recognition of translation-competent templates. The general contours and several important details of this process have become known only recently and represent the subject of this review.

Homologs of aquifex aeolicus protein-only RNase P are not the major RNase P activities in the archaea haloferax volcanii and methanosarcina mazei.

The mature 5'-ends of tRNAs are generated by RNase P in all domains of life. The ancient form of the enzyme is a ribonucleoprotein consisting of a catalytic RNA and one or more protein subunits. However, in the hyperthermophilic bacterium Aquifex aeolicus and close relatives, RNase P is a protein-only enzyme consisting of a single type of polypeptide (Aq_880, ~23 kDa). In many archaea, homologs of Aq_880 were identified (termed HARPs for Homologs of Aquifex RNase P) in addition to the RNA-based RNase P, raising the question about the functions of HARP and the classical RNase P in these archaea. Here we investigated HARPs from two euryarchaeotes, Haloferax volcanii and Methanosarcina mazei. Archaeal strains with HARP gene knockouts showed no growth phenotypes under standard conditions, temperature and salt stress (H. volcanii) or nitrogen deficiency (M. mazei). Recombinant H. volcanii and M. mazei HARPs were basically able to catalyse specific tRNA 5'-end maturation in vitro. Furthermore, M. mazei HARP was able to rescue growth of an Escherichia coli RNase P depletion strain with comparable efficiency as Aq_880, while H. volcanii HARP was unable to do so. In conclusion, both archaeal HARPs showed the capacity (in at least one functional assay) to act as RNases P. However, the ease to obtain knockouts of the singular HARP genes and the lack of growth phenotypes upon HARP gene deletion contrasts with the findings that the canonical RNase P RNA gene cannot be deleted in H. volcanii, and a knockdown of RNase P RNA in H. volcanii results in severe tRNA processing defects. We conclude that archaeal HARPs do not make a major contribution to global tRNA 5'-end maturation in archaea, but may well exert a specialised, yet unknown function in (t)RNA metabolism. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1109-1116, 2019.

DSP1 and DSP4 Act Synergistically in Small Nuclear RNA 3' End Maturation and Pollen Growth.

Small nuclear RNAs (snRNAs) play essential roles in spliceosome assembly and splicing. Most snRNAs are transcribed by the DNA-dependent RNA polymerase II (Pol II) and require 3'-end endonucleolytic cleavage. We have previously shown that the Arabidopsis (Arabidopsis thaliana) Defective in snRNA Processing 1 (DSP1) complex, composed of at least five subunits, is responsible for snRNA 3' maturation and is essential for plant development. Yet it remains unclear how DSP1 complex subunits act together to process snRNAs. Here, we show that DSP4, a member of the metallo-β-lactamase family, physically interacts with DSP1 through its β-Casp domain. Null dsp4-1 mutants have pleiotropic developmental defects, including impaired pollen development and reduced pre-snRNA transcription and 3' maturation, resembling the phenotype of the dsp1-1 mutant. Interestingly, dsp1-1 dsp4-1 double mutants exhibit complete male sterility and reduced pre-snRNA transcription and 3'-end maturation, unlike dsp1-1 or dsp4-1 In addition, Pol II occupancy at snRNA loci is lower in dsp1-1 dsp4-1 than in either single mutant. We also detected miscleaved pre-snRNAs in dsp1-1 dsp4-1, but not in dsp1-1 or dsp4-1 Taken together, these data reveal that DSP1 and DSP4 function is essential for pollen development, and that the two cooperatively promote pre-snRNA transcription and 3'-end processing efficiency and accuracy.

Structural insights into the 3′-end mRNA maturation machinery: Snapshot on polyadenylation signal recognition

Pre-mRNA 3′-end maturation is achieved by a mechanism requiring four different protein complexes assembled from approximately twenty factors. A global understanding of this essential process is still missing due to the inability to structurally characterize the entire complexes, even though structures of the isolated factors have been obtained. In this review, we summarize recent findings regarding the atomic description of one of the major players, the Cleavage and Polyadenylation Specificity Factor complex (CPSF in human, CPF in yeast). These data provide information on the architecture adopted by the major components of this complex, and on its capacity to recognize the polyadenylation signal sequence.

Major contribution of transcription initiation to 5’-end formation of mitochondrial steady-state transcripts in maize

ABSTRACTIn plant mitochondria, some steady-state transcripts contain primary 5ʹ ends derived from transcription initiation, while the others have processed 5ʹ termini generated by post-transcriptional processing. Differentiation and mapping of the primary and processed transcripts are important for unraveling the molecular mechanism(s) underlying transcription and transcript end maturation. However, previous efforts to systematically differentiate these two types of transcripts in plant mitochondria failed. At present, it is considered that the majority of mature mRNAs may have processed 5ʹ ends in Arabidopsis. Here, by combination of circular RT-PCR, quantitative RT-PCR, RNA 5ʹ-polyphosphatase treatment and Northern blot, we successfully discriminated and mapped the primary and processed transcripts in maize mitochondria. Among the thirty-five mature and eight precursor RNAs analyzed in this study, about one half (21/43) were found to have multiple isoforms. In total, seventy-seven steady-state transcripts were determined, and forty-seven of them had primary 5ʹ ends. Most transcription initiation sites (126/167) were downstream of a crTA-motif. These data suggested a major contribution of transcription initiation to 5ʹ-end formation of steady-state transcripts in maize mitochondria. Moreover, the mapping results revealed that mature RNA termini had largely been formed before trans-splicing, and C→U RNA editing was accompanied with trans-splicing and transcript end formation in maize mitochondria.

The Arabidopsis chloroplast RNase J displays both exo- and robust endonucleolytic activities.

Arabidopsis chloroplast RNase J displaces both exo- and endo-ribonucleolytic activities and contains a unique GT-1 DNA binding domain. Control of chloroplast gene expression is predominantly at the post-transcriptional level via the coordinated action of nuclear encoded ribonucleases and RNA-binding proteins. The 5' end maturation of mRNAs ascribed to the combined action of 5'→3' exoribonuclease and gene-specific RNA-binding proteins of the pentatricopeptide repeat family and others that impede the progression of this nuclease. The exo- and endoribonuclease RNase J, the only prokaryotic 5'→3' ribonuclease that is commonly present in bacteria, Archaea, as well as in the chloroplasts of higher plants and green algae, has been implicated in this process. Interestingly, in addition to the metalo-β-lactamase and β-CASP domains, RNase J of plants contains a conserved GT-1 domain that was previously characterized in transcription factors that function in light and stress responding genes. Here, we show that the Arabidopsis RNase J (AtRNase J), when analyzed in vitro with synthetic RNAs, displays both 5'→3' exonucleolytic activity, as well as robust endonucleolytic activity as compared to its bacterial homolog RNase J1 of Bacillus subtilis. AtRNase J degraded single-stranded RNA and DNA molecules but displays limited activity on double stranded RNA. The addition of three guanosines at the 5' end of the substrate significantly inhibited the degradation activity, indicating that the sequence and structure of the RNA substrate modulate the ribonucleolytic activity. Mutation of three amino acid in the catalytic reaction center significantly inhibited both the endonucleolytic and exonucleolytic degradation activities, while deletion of the carboxyl GT-1 domain that is unique to the plant RNAse J proteins, had a little or no significant effect. The robust endonucleolytic activity of AtRNase J suggests its involvement in the processing and degradation of RNA in the chloroplast.

Therapeutic Targeting of Telomere Diseases Via Novel Small Molecule Modulators of Non-Coding RNA Biogenesis

Germline mutations that impair telomere maintenance cause dyskeratosis congenita (DC), and predispose to hematologic diseases including bone marrow failure, myelodysplastic syndrome, and acute myeloid leukemia. Mutations in several DC-associated genes (DKC1, NOP10, NHP2, NAF1, PARN, TERC) act by disrupting steady-state levels of the essential non-coding RNA component of telomerase (TERC), which is limiting for telomerase activity and consequently self-renewal capacity in human stem cells. Ectopic expression of TERC RNA restores telomere length in cellular models of DC, but this approach poses major challenges for clinical translation. Based on insights from genetic discoveries in DC, we hypothesized that manipulating non-coding RNA pathways could be therapeutically useful to augment TERC in patients with telomere diseases. We and others recently demonstrated that PARN mutations cause DC via destabilization of nascent TERC RNA. Specifically, PARN is an RNA exonuclease that is required to remove post-transcriptionally added, non-genomically encoded adenosine residues - “oligo(A) tails” - that target TERC for destruction by the nuclear exosome (Fig. A). We subsequently identified the non-canonical poly(A) polymerase PAPD5 as the enzyme responsible for TERC oligo-adenylation and destabilization (Fig. A), and demonstrated that RNA interference of PAPD5 restored telomere length in DC patient cells. To further address this, we disrupted PAPD5 by CRISPR/Cas9-mediated genome editing in DC patient induced pluripotent stem cells (iPSCs) and heterologous human cell lines. We found that graded deficiencies in PAPD5 resulted in dose-dependent increases in TERC RNA and telomere length, and that PAPD5-null clones could be isolated and propagated indefinitely. These results provide genetic evidence of an unanticipated therapeutic window for PAPD5 inhibition, as a potential strategy to modulate TERC RNA levels.Pharmacological inhibitors of PAPD5 have not been identified. We therefore developed and executed a high-throughput functional screen to discover novel small molecule PAPD5 inhibitors. Of 100,055 compounds tested, we identified several hits that inhibited recombinant PAPD5 (rPAPD5), and triaged based on potency, specificity, and activity in orthogonal in vitro assays. Here we report on one of these novel compounds studied most extensively, BCH001. In vitro, BCH001 inhibited rPAPD5-mediated oligonucleotide extension, and was specific for rPAPD5 compared to other canonical and non-canonical poly-nucleotide polymerases. Differential scanning fluorimetry indicated direct binding of BCH001 to rPAPD5 that was ATP- and dose-dependent. We next used human cell-based assays to determine the impact of BCH001 on TERC RNA processing, telomere dynamics and the transcriptome. In PARN-mutant iPSC clones derived from patients with telomere diseases, we found that treatment with BCH001 in the 100 nM - 1 µM range reduced TERC RNA oligo-adenylation and increased steady state TERC RNA levels. Strikingly and consistently, treatment with BCH001 led to telomere elongation across multiple patient iPSCs tested (Fig. B), establishing a normalized telomere length set-point over weeks that was reversible with drug removal. RNA-Seq analysis demonstrated that only the non-coding small Cajal body RNA 13 (scaRNA13), which is known to be dependent on PARN for stability, was significantly differentially expressed between drug-treated and untreated PARN-mutant patient iPSCs. These data indicate minimal changes across the transcriptome at doses of BCH001 that are sufficient to alter TERC RNA levels and restore telomere length in DC patient iPSCs. We further found that BCH001 could shift the balance in favor of productive TERC RNA 3' end maturation across a range of human cell types, including primary fibroblasts, transformed cell lines, normal iPSCs, and iPSCs from patients with specific DC-associated DKC1 and TERC mutations.Taken together, these data provide genetic and pharmacological evidence that TERC RNA and telomere length can be controlled in human cells by modulation of the PARN/PAPD5 post-transcriptional regulatory axis. This has important therapeutic implications for the hematologic and non-hematologic manifestations of DC and related disorders, and possibly a broad range of human degenerative diseases. DisclosuresNo relevant conflicts of interest to declare.