RNA Processing Mechanisms Research Articles

The Synaptosome Associated Protein‐23 is Necessary for Skeletal Muscle Myogenesis

Alternative splicing is a post‐transcriptional RNA processing mechanism that occurs in more than 90% of human genes and allows for many proteins to arise from one gene. Membrane trafficking genes are regulated by alternative splicing during postnatal development1. The synaptosome associated protein‐23 (Snap23) is a member of the SNARE complex and is involved in the fusion of transport vesicles with the plasma membrane before cargo release2. We have shown that the Snap23 gene is regulated by alternative splicing. Exon 6, which contains 33 nucleotides, is skipped in fetal stages and is gradually included in adulthood. This transition is exclusive to striated muscles. SNAP23 is essential for mouse viability3; however, its role during muscle development is unclear. We hypothesize that SNAP23 is necessary for maintenance of muscle cell architecture and that alternative splicing is involved as a regulatory mechanism of muscle development.Murine C2C12 cell differentiation (myogenesis) replicates the Snap23 splicing transitions that we observed in muscle development. Thus, C2C12 cells are an appropriate model to investigate SNAP23 function and its regulation by alternative splicing. C2C12 cells were transfected with siRNAs to deplete SNAP23 protein expression in the undifferentiated stage and then differentiated for four days. Depletion of Snap23 led to a reduction in cell viability and fusion, indicating that Snap23 is required for myogenesis. Media collected from healthy differentiated cells (conditioned media) rescue the phenotype of Snap23 knock‐down cells. Immunofluorescence confirmed greater cell differentiation than those treated with standard differentiation media. This led us to hypothesize that SNAP23 mediates the secretion of a protein that is necessary for myogenesis. We coated plates with various concentrations of gelatin and observed that gelatin‐coated plates also restored cell differentiation. We are currently investigating fibronectin as a potential important protein, as it is a major component of gelatin and interacts with integrins on the plasma membrane.In parallel, to study the implications of alternative splicing on Snap23 pre‐mRNA, we used morpholino antisense oligonucleotides (MO) to redirect endogenous splicing decisions. Cells treated with Snap23‐MO expressed exclusively the short isoform throughout myogenesis while control cells express the long isoform in differentiated stages. In Snap23‐MO treated cells, we observed more myotubes with greater nuclei per myotube compared to the controls, suggesting that Snap23 splicing contributes to cell fusion and thus myogenesis.In conclusion, our data suggest that SNAP23 and its regulation by alternative splicing is involved in the fusion of myoblasts to form multinucleated myotubes. We propose that SNAP23 mediates the secretion of proteins necessary for myogenesis. Ongoing studies are directed to elucidate the molecular mechanisms behind the observed phenotypes.Support or Funding InformationNIH/NIGMS award (R25 GM089569) (to J. Gamarra UNC‐Chapel Hill PREP Scholar) and start up funds from UNC‐Chapel Hill (to J. Giudice)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Integrator is a key component of human telomerase RNA biogenesis

Telomeres are special DNA-protein structures that are located at the ends of linear eukaryotic chromosomes. The telomere length determines the proliferation potential of cells. Telomerase is a key component of the telomere length maintenance system. While telomerase is inactive in the majority of somatic cells, its activity determines the clonogenic potential of stem cells as a resource for tissue and organism regeneration. Reactivation of telomerase occurs during the process of immortalization in the majority of cancer cells. Telomerase is a ribonucleoprotein that contains telomerase reverse transcriptase and telomerase RNA components. The RNA processing mechanism of telomerase involves exosome trimming or degradation of the primary precursor. Recent data provide evidence that the competition between the processing and decay of telomerase RNA may regulate the amount of RNA at the physiological level. We show that termination of human telomerase RNA transcription is dependent on its promoter, which engages with the multisubunit complex Integrator to interact with RNA polymerase II and terminate transcription of the human telomerase RNA gene followed by further processing.

Transcriptome, proteome and draft genome of Euglena gracilis

BackgroundPhotosynthetic euglenids are major contributors to fresh water ecosystems. Euglena gracilis in particular has noted metabolic flexibility, reflected by an ability to thrive in a range of harsh environments. E. gracilis has been a popular model organism and of considerable biotechnological interest, but the absence of a gene catalogue has hampered both basic research and translational efforts.ResultsWe report a detailed transcriptome and partial genome for E. gracilis Z1. The nuclear genome is estimated to be around 500 Mb in size, and the transcriptome encodes over 36,000 proteins and the genome possesses less than 1% coding sequence. Annotation of coding sequences indicates a highly sophisticated endomembrane system, RNA processing mechanisms and nuclear genome contributions from several photosynthetic lineages. Multiple gene families, including likely signal transduction components, have been massively expanded. Alterations in protein abundance are controlled post-transcriptionally between light and dark conditions, surprisingly similar to trypanosomatids.ConclusionsOur data provide evidence that a range of photosynthetic eukaryotes contributed to the Euglena nuclear genome, evidence in support of the ‘shopping bag’ hypothesis for plastid acquisition. We also suggest that euglenids possess unique regulatory mechanisms for achieving extreme adaptability, through mechanisms of paralog expansion and gene acquisition.

Homology-independent multiallelic disruption via CRISPR/Cas9-based knock-in yields distinct functional outcomes in human cells

BackgroundCultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes.ResultsExemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1−/− cells and attenuated cytokine-induced cell death in FAT10−/− clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIP transcripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIP transcripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones.ConclusionMultiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partially functional transcripts through diverse DNA and RNA processing mechanisms.

The emerging role of PTBP1 in human cancer: novel prognostic factor in non-muscle invasive bladder cancer.

Alternative splicing is a sophisticated RNA processing mechanism that expands the coding potential of the genome and the proteomic complexity in higher eukaryotes. Splicing requires the recognition of the intron/exon boundaries by a large ribonucleoprotein complex, named the spliceosome, followed by intron excision and ligation of exons to yield the messenger RNA (mRNA) (1).

Subcellular Compartmentation of Alternatively Spliced Transcripts Defines SERINE/ARGININE-RICH PROTEIN30 Expression.

Alternative splicing (AS) is prevalent in higher eukaryotes, and generation of different AS variants is tightly regulated. Widespread AS occurs in response to altered light conditions and plays a critical role in seedling photomorphogenesis, but despite its frequency and effect on plant development, the functional role of AS remains unknown for most splicing variants. Here, we characterized the light-dependent AS variants of the gene encoding the splicing regulator Ser/Arg-rich protein SR30 in Arabidopsis (Arabidopsis thaliana). We demonstrated that the splicing variant SR30.2, which is predominantly produced in darkness, is enriched within the nucleus and strongly depleted from ribosomes. Light-induced AS from a downstream 3' splice site gives rise to SR30.1, which is exported to the cytosol and translated, coinciding with SR30 protein accumulation upon seedling illumination. Constitutive expression of SR30.1 and SR30.2 fused to fluorescent proteins revealed their identical subcellular localization in the nucleoplasm and nuclear speckles. Furthermore, expression of either variant shifted splicing of a genomic SR30 reporter toward SR30.2, suggesting that an autoregulatory feedback loop affects SR30 splicing. We provide evidence that SR30.2 can be further spliced and, unlike SR30.2, the resulting cassette exon variant SR30.3 is sensitive to nonsense-mediated decay. Our work delivers insight into the complex and compartmentalized RNA processing mechanisms that control the expression of the splicing regulator SR30 in a light-dependent manner.

Caloric Restriction Engages Hepatic RNA Processing Mechanisms in Rhesus Monkeys

Caloric restriction (CR) extends lifespan and delays the onset of age-related disorders in diverse species. Metabolic regulatory pathways have been implicated in the mechanisms of CR, but the molecular details have not been elucidated. Here, we show that CR engages RNA processing of genes associated with a highly integrated reprogramming of hepatic metabolism. We conducted molecular profiling of liver biopsies collected from adult male rhesus monkeys (Macaca mulatta) at baseline and after 2 years on control or CR (30% restricted) diet. Quantitation of over 20,000 molecules from the hepatic transcriptome, proteome, and metabolome indicated that metabolism and RNA processing are major features of the response to CR. Predictive models identified lipid, branched-chain amino acid, and short-chain carbon metabolic pathways, with alternate transcript use for over half of the genes in the CR network. Weconclude that RNA-based mechanisms are central tothe CR response and integral in metabolic reprogramming.

Insights into the role of ribonuclease 4 polymorphisms in amyotrophic lateral sclerosis

Mutations in certain genes of the Ribonuclease (RNASE) superfamily can cause amyotrophic lateral sclerosis (ALS) through altered RNA processing mechanisms. About 30 of these missense mutations in RNASE5/ANG gene have already been reported in ALS patients. In another gene of the ribonuclease superfamily, ribonuclease 4 (RNASE4), missense mutations and single nucleotide polymorphisms have been identified in patients suffering from ALS. However, their plausible molecular mechanisms of association with ALS are not known. Here, we present the molecular mechanisms of RNASE4 polymorphisms with ALS using all-atom molecular dynamics (MD) simulations followed by functional assay experiments. As most ALS causing mutations in RNASE superfamily proteins affect either the ribonucleolytic or nuclear translocation activity, we examined these functional properties of wild-type and known RNASE4 variants, R10W, A98V, E48D and V75I, using MD simulations. Our simulation predicted that these variants would retain nuclear translocation activity and that E48D would exhibit loss of ribonucleolytic activity, which was subsequently validated by ribonucleolytic assay. Our results give a mechanistic insight into the association of RNASE4 polymorphisms with ALS and show that E48D-RNASE4 would probably be deleterious and cause ALS in individuals harbouring this polymorphism.

Advances in Transcriptomics of Plants.

The current global population of 7.3 billion is estimated to reach 9.7 billion in the year 2050. Rapid population growth is driving up global food demand. Additionally, global climate change, environmental degradation, drought, emerging diseases, and salty soils are the current threats to global food security. In order to mitigate the adverse effects of these diverse agricultural productivity constraints and enhance crop yield and stress-tolerance in plants, we need to go beyond traditional and molecular plant breeding. The powerful new tools for genome editing, Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)/Cas systems (CRISPR-Cas9), have been hailed as a quantum leap forward in the development of stress-resistant plants. Plant breeding techniques, however, have several drawbacks. Hence, identification of transcriptional regulatory elements and deciphering mechanisms underlying transcriptional regulation are crucial to avoiding unintended consequences in modified crop plants, which could ultimately have negative impacts on human health. RNA splicing as an essential regulated post-transcriptional process, alternative polyadenylation as an RNA-processing mechanism, along with non-coding RNAs (microRNAs, small interfering RNAs and long non-coding RNAs) have been identified as major players in gene regulation. In this chapter, we highlight new findings on the essential roles of alternative splicing and alternative polyadenylation in plant development and response to biotic and abiotic stresses. We also discuss biogenesis and the functions of microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants and recent advances in our knowledge of the roles of miRNAs and siRNAs in plant stress response. Graphical Abstract.

Transgene-Like Animal Models Using Intronic MicroRNAs.

Transgenic animal models are valuable tools for testing gene functions and drug mechanisms in vivo. They are also the best similitude for a human body for etiological and pathological research of diseases. All pharmaceutically developed medicines must be proven to be safe and effective in animals before approval by the Food and Drug Administration(FDA) to be used in clinical trials. To this end, the transgenic animal models of diseases serve as the front line of drug evaluation. However, there is currently no transgenic animal model for microRNA (miRNA)-related research. MiRNAs, small single-stranded regulatory RNAs capable of silencing intracellular gene transcripts (mRNAs) that contain either complete or partial complementarity to the miRNA, are useful for the design of new therapies against cancer polymorphism and viral mutation. Recently, varieties of natural miRNAs have been found to be derived from hairpin-like RNA precursors in almost all eukaryotes, including yeast (Schizosaccharomyces pombe), plant (Arabidopsis spp.), nematode (Caenorhabditis elegans), fly (Drosophila melanogaster), fish, mouse and human, involving intracellular defense against viral infections and regulation of certain gene expressions during development. To facilitate the miRNA research in vivo, we have developed a state-of-the-art transgenic strategy for silencing specific genes in zebrafish, chicken, and mouse, using intronic miRNAs. By the insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, we have found that mature miRNAs were successfully transcribed by RNA polymerases type II (Pol-II), coexpressed with the encoding gene transcripts, and excised out of the encoding gene transcripts by intracellular RNA splicing and processing mechanisms. In conjunction with retroviral transfection, the designed hairpin-like pre-miRNA construct has also been placed in the intron regions of a cellular gene for tissue-specific expression, specifically regulated by the gene promoterof interest. Because the retroviral vectors are integrated into the genome of its host cells,we can select and propagate the most effective transgenic animals to form a stable model line for further research. Here, we have shown for the first time that transgene-like animal models were generated using the intronic miRNAexpression system reported previously, which has been proven to be useful for studying miRNA function as well as the relatedgeneregulation in vivo.

Abstract B172: Identification of a novel RNA processing mechanism of intronic readthrough to a transcriptional stop leading to truncated transcript expression, including FANCI and ATM, upon CDK12 inhibition

Abstract Cyclin-dependent kinase 12 (CDK12), a key regulator of transcriptional processing along with its binding partner cyclin K (CCNK), has been implicated in the modulation of genes involved with the DNA damage response (DDR) including, but not limited to, homologous recombination repair (HRR). However, the exact mechanism by which CDK12 is having this effect has been unclear. To identify differentially expressed transcripts between wild-type and mutant CDK12 that may indicate alternative or mis-splicing events and help understand the mechanism of CDK12 loss of function, we analyzed RNA-Seq data from TCGA ovarian cancer comparing transcripts in loss of function mutated CDK12 (~3% of ovarian) vs wild-type samples. We identified a number of transcripts that were at least twofold higher in CDK12 mutant vs wild-type samples. Deeper analysis showed an enrichment in truncated transcripts, a subset of which may arise from readthrough into an intron containing a transcriptional stop, including the DDR genes FANCI and ATM. This potential mechanism by which CDK12 may regulate DDR genes was tested in vitro using Taqman probes upstream and downstream of the introns with the stop signal for both ATM and FANCI. To validate the differential expression of FANCI and ATM transcripts observed in TCGA samples, we treated the ovarian cells lines OVCAR8 and OV90 with CDK12 siRNA for 72hrs. CDK12 knockdown resulted in truncated transcript expression and a reduction in the full-length transcripts of DDR genes FANCI and ATM. Reduction in levels of full-length transcripts also leads to decreased levels of FANCI and ATM protein. Knockdown of the closely related homolog CDK13 by siRNA did not lead to expression of truncated FANCI and ATM transcripts. Sensitization to olaparib was observed for CDK12 but not CDK13 siRNA treated ovarian cells in a 14-day clonogenic assay indicating a loss of HRR proficiency due to CDK12 depletion. A biochemical screen was developed and a synthetic chemistry plan initiated to identify CDK12 selective probe compounds to try to confirm the target biology, DDR gene modulation, and RNA processing effects seen with siRNA. Potent and selective inhibitors of CDK12/13 were identified and used to treat ovarian cancer cells. Differential expression of full-length vs truncated FANCI and ATM transcripts was seen by 4-8 hrs with CDK12/13 selective compounds. However, at higher concentrations and longer time points, effects on general transcription were observed as indicated by a decrease in full-length ATM and FANCI, potent effects on p-Ser2 of RNApolII, and growth inhibition in a 48-hr proliferation assay. The small-molecule inhibitors partially recapitulate the RNA processing effects on ATM and FANCI; however, effect on CDK13 or the retention of CCNK in an inactive complex may lead to broader transcriptional effects. To our knowledge, these are the first data linking CDK12 inhibition to a RNA processing mechanism of intronic readthrough to a transcriptional stop leading to modulation of a subset of transcripts, including the DDR genes ATM and FANCI. Citation Format: Christopher Denz, Jeffrey Johannes, Yi Yao, Meghana Kulkarni, Austin Dulak, Nin Guan, Nancy Su, Michelle Lamb, Stephen Fawell, Sylvie Guichard. Identification of a novel RNA processing mechanism of intronic readthrough to a transcriptional stop leading to truncated transcript expression, including FANCI and ATM, upon CDK12 inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B172.

On the Possibility of an Early Evolutionary Origin for the Spliced Leader Trans-Splicing.

Trans-splicing is a process by which 5'- and 3'-ends of two pre-RNA molecules transcribed from different sites of the genome can be joined together to form a single RNA molecule. The spliced leader (SL) trans-splicing is mediated by the spliceosome and it allows the replacement of 5'-end of pre-mRNA by 5'(SL)-end of SL-RNA. This form of splicing has been observed in many phylogenetically unrelated eukaryotes. Either the SL trans-splicing (SLTS) originated in the last eukaryotic common ancestor (LECA) (or even earlier) and it was lost in most eukaryotic lineages, or this mechanism of RNA processing evolved several times independently in various unrelated eukaryotic taxa. The bioinformatic comparisons of SL-RNAs from various eukaryotic taxonomic groups have revealed the similarities of secondary structures of most SL-RNAs and a relative conservation of their splice sites (SSs) and Sm-binding sites (SmBSs). We propose that such structural and functional similarities of SL-RNAs are unlikely to have evolved repeatedly many times. Hence, we favor the scenario of an early evolutionary origin for the SLTS and multiple losses of SL-RNAs in various eukaryotic lineages.

Abstract 3181: A novel topoisomerase IIα 90 kDa isoform in etoposide resistant human leukemia K562 cells produced as a result of alternative RNA processing

Abstract DNA topoisomerase IIα (TOP2α) is a prominent target for anticancer drugs whose clinical efficacy is often limited by chemoresistance. We previously characterized acquired resistance to etoposide (VP-16) in a cloned human K562 leukemia cell line, K/VP.5, containing reduced TOP2α. In the present study, using an antibody specific for the amino-terminus of TOP2α, immunoassays indicated the existence of two TOP2α isoforms, 170 and 90 kDa, present in K562 leukemia cells and in the etoposide resistant K/VP.5 cells. TOP2α/90 expression was dramatically increased in etoposide-resistant K/VP.5 compared to parental K562 cells. We hypothesized that TOP2α/90 was the translation product of novel alternatively processed pre-mRNA, confirmed by 3’-RACE, PCR, and sequencing. TOP2α/90 mRNA includes retained intron 19 which harbors an in-frame stop codon, and two consensus poly(A) sites. The processed transcript is polyadenylated. TOP2α/90 mRNA encodes a 90,076 Da translation product missing the carboxyl-terminal 770 amino acids of TOP2α/170, replaced by 25 unique amino acids through translation of the exon 19/intron 19 ‘readthrough’. Immunoassays, utilizing antisera raised against these unique amino acids, confirmed that TOP2α/90 is expressed in both cell types, with overexpression in K/VP.5 cells. Immunodetection of Complex of Enzyme-to-DNA (ICE) and single cell gel electrophoresis (Comet) assays demonstrated that K562 cells transfected with a TOP2α/90 expression plasmid, exhibited reduced etoposide-mediated TOP2α-DNA covalent complexes and decreased etoposide-induced DNA damage, respectively, compared to similarly treated K562 cells transfected with empty vector. Since TOP2α/90 lacks the active site tyrosine (Tyr805) of full length TOP2α, these results strongly suggest that TOP2α/90 exhibits dominant-negative properties. . In separate studies the TOP2α/90 mRNA splice variant was found to be expressed in most human tissues suggesting that this novel protein isoform may play a role in both intrinsic chemosensitivity as well as in acquired resistance. Further studies are underway to characterize the mechanism(s) by which TOP2α/90 plays a role in acquired resistance to etoposide and other TOP2α targeting agents. In addition, future studies will be directed to examine the RNA processing mechanism(s) operational that suppress intron 19 splicing in TOP2α pre-mRNA. Citation Format: Ragu Kanagasabai, Lucas Serdar, Soumendrakrishna Karmahapatra, Corey A. Kientz, Mary K. Ritke, Terry S. Elton, Jack C. Yalowich. A novel topoisomerase IIα 90 kDa isoform in etoposide resistant human leukemia K562 cells produced as a result of alternative RNA processing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3181. doi:10.1158/1538-7445.AM2017-3181

The Canonical Poly (A) Polymerase PAP1 Polyadenylates Non-Coding RNAs and Is Essential for snoRNA Biogenesis in Trypanosoma brucei

The parasite Trypanosoma brucei is the causative agent of African sleeping sickness and is known for its unique RNA processing mechanisms that are common to all the kinetoplastidea including Leishmania and Trypanosoma cruzi. Trypanosomes possess two canonical RNA poly (A) polymerases (PAPs) termed PAP1 and PAP2. PAP1 is encoded by one of the only two genes harboring cis-spliced introns in this organism, and its function is currently unknown. In trypanosomes, all mRNAs, and non-coding RNAs such as small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs), undergo trans-splicing and polyadenylation. Here, we show that the function of PAP1, which is located in the nucleus, is to polyadenylate non-coding RNAs, which undergo trans-splicing and polyadenylation. Major substrates of PAP1 are the snoRNAs and lncRNAs. Under the silencing of either PAP1 or PAP2, the level of snoRNAs is reduced. The dual polyadenylation of snoRNA intermediates is carried out by both PAP2 and PAP1 and requires the factors essential for the polyadenylation of mRNAs. The dual polyadenylation of the precursor snoRNAs by PAPs may function to recruit the machinery essential for snoRNA processing.

Alternative splicing and the evolution of phenotypic novelty.

Alternative splicing, a mechanism of post-transcriptional RNA processing whereby a single gene can encode multiple distinct transcripts, has been proposed to underlie morphological innovations in multicellular organisms. Genes with developmental functions are enriched for alternative splicing events, suggestive of a contribution of alternative splicing to developmental programmes. The role of alternative splicing as a source of transcript diversification has previously been compared to that of gene duplication, with the relationship between the two extensively explored. Alternative splicing is reduced following gene duplication with the retention of duplicate copies higher for genes which were alternatively spliced prior to duplication. Furthermore, and unlike the case for overall gene number, the proportion of alternatively spliced genes has also increased in line with the evolutionary diversification of cell types, suggesting alternative splicing may contribute to the complexity of developmental programmes. Together these observations suggest a prominent role for alternative splicing as a source of functional innovation. However, it is unknown whether the proliferation of alternative splicing events indeed reflects a functional expansion of the transcriptome or instead results from weaker selection acting on larger species, which tend to have a higher number of cell types and lower population sizes.This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'.

Functional Genomics Identifies Tis21-Dependent Mechanisms and Putative Cancer Drug Targets Underlying Medulloblastoma Shh-Type Development.

We have recently generated a novel medulloblastoma (MB) mouse model with activation of the Shh pathway and lacking the MB suppressor Tis21 (Patched1+/−/Tis21KO). Its main phenotype is a defect of migration of the cerebellar granule precursor cells (GCPs). By genomic analysis of GCPs in vivo, we identified as drug target and major responsible of this defect the down-regulation of the promigratory chemokine Cxcl3. Consequently, the GCPs remain longer in the cerebellum proliferative area, and the MB frequency is enhanced. Here, we further analyzed the genes deregulated in a Tis21-dependent manner (Patched1+/−/Tis21 wild-type vs. Ptch1+/−/Tis21 knockout), among which are a number of down-regulated tumor inhibitors and up-regulated tumor facilitators, focusing on pathways potentially involved in the tumorigenesis and on putative new drug targets. The data analysis using bioinformatic tools revealed: (i) a link between the Shh signaling and the Tis21-dependent impairment of the GCPs migration, through a Shh-dependent deregulation of the clathrin-mediated chemotaxis operating in the primary cilium through the Cxcl3-Cxcr2 axis; (ii) a possible lineage shift of Shh-type GCPs toward retinal precursor phenotype, i.e., the neural cell type involved in group 3 MB; (iii) the identification of a subset of putative drug targets for MB, involved, among the others, in the regulation of Hippo signaling and centrosome assembly. Finally, our findings define also the role of Tis21 in the regulation of gene expression, through epigenetic and RNA processing mechanisms, influencing the fate of the GCPs.

식물의 긴비암호화 RNA들의 생물학적 기능

차세대 염기서열 분석기술의 발달로 대량의 전사수준의 단위체들이 발견되었는데, 이것들 중 대부분의 전사체들은 비암호화 RNA들이다. 이들 중 긴 비암호화 RNA (lncRNA)들은 200개 이상의 뉴클레오티드를 가지며 단백질로 번역이 되지 않는 기능적 RNA 분자들이다. 식물의 lncRNA들은 RNA Pol II, Pol III, Pol IV, Pol V에 의해 전사체가 만들어지고, 전사 후 이들 lncRNA들은 추가적인 splicing과 polyadenylation 과정이 일어난다. 식물의 lncRNA들은 그 발현수준이 매우 낮고, 조직 특이적으로 발현 되지만, 이들 lncRNA들은 외부자극에 의해 강한 발현이 유도된다. 환경스트레스를 포함한 각기 다른 외부자극에 의해 많은 식물 lncRNA들의 발현이 유도되었기 때문에 이들 lncRNA들은 식물의 다양한 생물학적 기능과 식물의 생장발달 과정에 있어 새로운 조절 인자로 고려되고 있다. 특히 후성유전학적인 유전자 억제, 크로마틴 변형, 타겟모방, 광형태형성, 단백질 재배치, 환경스트레스 반응, 병원균 감염등에 관련하여 기능을 한다. 또한 어떤 lncRNA들은 short RNA들의 전구체로 역할도 한다. 최근 식물에서 많은 lncRNA들이 분리 동정되었지만, 이들 lncRNA들의 생리학적인 기능에 대한 현재의 이해는 여전히 제한적이고, 이들의 세부적인 조절 메커니즘 연구는 지속적으로 이루어져야 할 것이다. 이 총설에서는 식물 lncRNA들의 생합성 및 조절 메커니즘과 현재까지 밝혀진 분자수준에서의 중요한 기능들을 요약 정리하였다. With the development of next generation sequencing (NGS), large numbers of transcriptional molecules have been discovered. Most transcripts are non -coding RNAs (ncRNAs). Among them, long non-coding RNAs (lncRNAs) with more than 200 nucleotides represent functional RNA molecule that will not be translated into protein. In plants, lncRNAs are transcribed by RNA polymerase II (Pol II) or Pol III, Pol VI and Pol V. After transcription of these lncRNAs, more RNA processing mechanisms such as splicing and polyadenylation occurs. The expression of plant lncRNAs is very low and is tissue specific. However, these lncRNAs are strongly induced by specific external stimuli. Because different external stimuli including environmental stresses induce a large number of plant lncRNAs, these lncRNAs have been gradually considered as new regulatory factors of various biological and development processes such as epigenetic repression, chromatin modification, target mimicry, photomorphogenesis, protein relocalization, environmental stress response, pathogen infection in plants. Moreover, some lncRNAs act as precursor of short RNAs. Although a large number of lncRNAs have been predicted and identified in plants, our current understanding of the biological function of these lncRNAs is still limited and their detailed regulatory mechanisms should be elucidated continuously. Here, we reviewed the biogenesis and regulation mechanisms of lncRNAs and summarized the molecular functions unraveled in plants.

Alternative polyadenylation of mRNA precursors.

Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3' termini on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA exhibits tissue specificity and is important for cell proliferation and differentiation. In this Review, we discuss the roles of APA in diverse cellular processes, including mRNA metabolism, protein diversification and protein localization, and more generally in gene regulation. We also discuss the molecular mechanisms underlying APA, such as variation in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation.

Genome-wide analysis of small nucleolar RNAs of Leishmania major reveals a rich repertoire of RNAs involved in modification and processing of rRNA

Trypanosomatids are protozoan parasites and the causative agent of infamous infectious diseases. These organisms regulate their gene expression mainly at the post-transcriptional level and possess characteristic RNA processing mechanisms. In this study, we analyzed the complete repertoire of Leishmania major small nucleolar (snoRNA) RNAs by performing RNA-seq analysis on RNAs that were affinity-purified using the C/D snoRNA core protein, SNU13, and the H/ACA core protein, NHP2. This study revealed a large collection of C/D and H/ACA snoRNAs, organized in gene clusters generally containing both snoRNA types. Abundant snoRNAs were identified and predicted to guide trypanosome-specific rRNA cleavages. The repertoire of snoRNAs was compared to that of the closely related Trypanosoma brucei, and 80% of both C/D and H/ACA molecules were found to have functional homologues. The comparative analyses elucidated how snoRNAs evolved to generate molecules with analogous functions in both species. Interestingly, H/ACA RNAs have great flexibility in their ability to guide modifications, and several of the RNA species can guide more than one modification, compensating for the presence of single hairpin H/ACA snoRNA in these organisms. Placing the predicted modifications on the rRNA secondary structure revealed hypermodification regions mostly in domains which are modified in other eukaryotes, in addition to trypanosome-specific modifications.

Cellulosome stoichiometry in Clostridium cellulolyticum is regulated by selective RNA processing and stabilization.

The mechanism, physiological relevance and evolutionary implication of selective RNA processing and stabilization (SRPS) remain elusive. Here we report the genome-wide maps of transcriptional start sites (TSs) and post-transcriptional processed sites (PSs) for Clostridium cellulolyticum. The PS-associated genes are preferably associated with subunits of heteromultimeric protein complexes, and the intergenic PSs (iPSs) are enriched in operons exhibiting highly skewed transcript-abundance landscape. Stem-loop structures associated with those iPSs located at 3′ termini of highly transcribed genes exhibit folding free energy negatively correlated with transcript-abundance ratio of flanking genes. In the cellulosome-encoding cip-cel operon, iPSs and stem-loops precisely regulate structure and abundance of the subunit-encoding transcripts processed from a primary polycistronic RNA, quantitatively specifying cellulosome stoichiometry. Moreover, cellulosome evolution is shaped by the number, position and biophysical nature of TSs, iPSs and stem-loops. Our findings unveil a genome-wide RNA-encoded strategy controlling in vivo stoichiometry of protein complexes.