Polyadenylated Transcripts Research Articles

The MTR4/hnRNPK complex surveils aberrant polyadenylated RNAs with multiple exons

RNA surveillance systems degrade aberrant RNAs that result from defective transcriptional termination, splicing, and polyadenylation. Defective RNAs in the nucleus are recognized by RNA-binding proteins and MTR4, and are degraded by the RNA exosome complex. Here, we detect aberrant RNAs in MTR4-depleted cells using long-read direct RNA sequencing and 3′ sequencing. MTR4 destabilizes intronic polyadenylated transcripts generated by transcriptional read-through over one or more exons, termed 3′ eXtended Transcripts (3XTs). MTR4 also associates with hnRNPK, which recognizes 3XTs with multiple exons. Moreover, the aberrant protein translated from KCTD13 3XT is a target of the hnRNPK-MTR4-RNA exosome pathway and forms aberrant condensates, which we name KCTD13 3eXtended Transcript-derived protein (KeXT) bodies. Our results suggest that RNA surveillance in human cells inhibits the formation of condensates of a defective polyadenylated transcript-derived protein.

RNA infrastructure profiling illuminates transcriptome structure in crowded spaces.

RNAs fold into compact structures and undergo protein interactions in cells. These occluded environments can block reagents that probe the underlying RNAs. Probes that can analyze structure in crowded settings can shed light on RNA biology. Here, we employ 2'-OH-reactive probes that are small enough to access folded RNA structure underlying close molecular contacts within cells, providing considerably broader coverage for intracellular RNA structural analysis. The data are analyzed first with well-characterized human ribosomal RNAs and then applied transcriptome-wide to polyadenylated transcripts. The smallest probe acetylimidazole (AcIm) yields 80% greater structural coverage than larger conventional reagent NAIN3, providing enhanced structural information in hundreds of transcripts. The acetyl probe also provides superior signals for identifying m6A modification sites in transcripts, particularly in sites that are inaccessible to a standard probe. Our strategy enables profiling RNA infrastructure, enhancing analysis of transcriptome structure, modification, and intracellular interactions, especially in spatially crowded settings.

NERD-seq: a novel approach of Nanopore direct RNA sequencing that expands representation of non-coding RNAs

Non-coding RNAs (ncRNAs) are frequently documented RNA modification substrates. Nanopore Technologies enables the direct sequencing of RNAs and the detection of modified nucleobases. Ordinarily, direct RNA sequencing uses polyadenylation selection, studying primarily mRNA gene expression. Here, we present NERD-seq, which enables detection of multiple non-coding RNAs, excluded by the standard approach, alongside natively polyadenylated transcripts. Using neural tissues as a proof of principle, we show that NERD-seq expands representation of frequently modified non-coding RNAs, such as snoRNAs, snRNAs, scRNAs, srpRNAs, tRNAs, and rRFs. NERD-seq represents an RNA-seq approach to simultaneously study mRNA and ncRNA epitranscriptomes in brain tissues and beyond.

Description of Bacterial RNA Transcripts Detected in Mycobacterium tuberculosis - Infected Cells from Peripheral Human Granulomas using Single Cell RNA Sequencing.

Mycobacterium tuberculosis (Mtb) remains a global human health threat and a significant cause of human morbidity and mortality. We document here the capture of Mtb transcripts in libraries designed to amplify eukaryotic mRNA. These reads are often considered spurious or nuisance and are rarely investigated. Because of early literature suggesting the possible presence of polyadenylated transcripts in Mtb RNA, we included the H37Rv Mtb reference genome when assembling scRNA seq libraries from fine needle aspirate samples from patients presenting at the TB clinic, Port Moresby General Hospital, Papua New Guinea. We used 10X Genomics single-cell RNA sequencing transcriptomics pipeline, which initiates mRNA amplification with poly-T primers on ~30-micron beads designed to capture, in this case, human mRNA associated with individual cells in the clinical samples. Utilizing the 10X Genomics Cell Ranger tool to align sequencing reads, we consistently detected bacterial small and large ribosomal subunit RNA sequences (rrs and rrl, respectively) and other bacterial gene transcripts in the cell culture and patient samples. We interpret Mtb reads associated with the host cell's unique molecular identifier (UMI) and transcriptome to indicate infection of that individual host cell. The Mtb transcripts detected showed frequent sequence variation from the reference genome, with greater than 90% of the rrs or rrl reads from many clinical samples having at least 1 sequence difference compared to the H37Rv reference genome. The data presented includes only bacterial sequences from patients with TB infections that were confirmed by the hospital pathology lab using acid-fast microscopy and/or GeneXpert analysis. The repeated, non-random nature of the sequence variations detected in Mtb rrs and rrl transcripts from multiple patients, suggests that, even though this appears to be a stochastic process, there is possibly some selective pressure that limits the types and locations of sequence variation allowed. The variation does not appear to be entirely artefactual, and it is hypothesized that it could represent an additional mechanism of adaptation to enhance bacterial fitness against host defenses or chemotherapy.

Productive mRNA Chromatin Escape is Promoted by PRMT5 Methylation of SNRPB.

Protein Arginine Methyltransferase 5 (PRMT5) regulates RNA splicing and transcription by symmetric dimethylation of arginine residues (Rme2s/SDMA) in many RNA binding proteins. However, the mechanism by which PRMT5 couples splicing to transcriptional output is unknown. Here, we demonstrate that a major function of PRMT5 activity is to promote chromatin escape of a novel, large class of mRNAs that we term Genomically Retained Incompletely Processed Polyadenylated Transcripts (GRIPPs). Using nascent and total transcriptomics, spike-in controlled fractionated cell transcriptomics, and total and fractionated cell proteomics, we show that PRMT5 inhibition and knockdown of the PRMT5 SNRP (Sm protein) adapter protein pICln (CLNS1A) -but not type I PRMT inhibition-leads to gross detention of mRNA, SNRPB, and SNRPD3 proteins on chromatin. Compared to most transcripts, these chromatin-trapped polyadenylated RNA transcripts have more introns, are spliced slower, and are enriched in detained introns. Using a combination of PRMT5 inhibition and inducible isogenic wildtype and arginine-mutant SNRPB, we show that arginine methylation of these snRNPs is critical for mediating their homeostatic chromatin and RNA interactions. Overall, we conclude that a major role for PRMT5 is in controlling transcript processing and splicing completion to promote chromatin escape and subsequent nuclear export.

Nanoparticle delivery of Tat synergizes with classical latency reversal agents to express HIV antigen targets.

Limited cellular levels of the HIV transcriptional activator Tat are one contributor to proviral latency that might be targeted in HIV cure strategies. We recently demonstrated that lipid nanoparticles containing HIV tat mRNA induce HIV expression in primary CD4 T cells. Here, we sought to further characterize tat mRNA in the context of several benchmark latency reversal agents (LRAs), including inhibitor of apoptosis protein antagonists (IAPi), bromodomain and extra-Terminal motif inhibitors (BETi), and histone deacetylase inhibitors (HDACi). tat mRNA reversed latency across several different cell line models of HIV latency, an effect dependent on the TAR hairpin loop. Synergistic enhancement of tat mRNA activity was observed with IAPi, HDACi, and BETi, albeit to variable degrees. In primary CD4 T cells from durably suppressed people with HIV, tat mRNA profoundly increased the frequencies of elongated, multiply-spliced, and polyadenylated HIV transcripts, while having a lesser impact on TAR transcript frequencies. tat mRNAs alone resulted in variable HIV p24 protein induction across donors. However, tat mRNA in combination with IAPi, BETi, or HDACi markedly enhanced HIV RNA and protein expression without overt cytotoxicity or cellular activation. Notably, combination regimens approached or in some cases exceeded the latency reversal activity of maximal mitogenic T cell stimulation. Higher levels of tat mRNA-driven HIV p24 induction were observed in donors with larger mitogen-inducible HIV reservoirs, and expression increased with prolonged exposure time. Combination LRA strategies employing both small molecule inhibitors and Tat delivered to CD4 T cells are a promising approach to effectively target the HIV reservoir.

Fast and flexible profiling of chromatin accessibility and total RNA expression in single nuclei using Microwell-seq3

Single cell chromatin accessibility profiling and transcriptome sequencing are the most widely used technologies for single-cell genomics. Here, we present Microwell-seq3, a high-throughput and facile platform for high-sensitivity single-nucleus chromatin accessibility or full-length transcriptome profiling. The method combines a preindexing strategy and a penetrable chip-in-a-tube for single nucleus loading and DNA amplification and therefore does not require specialized equipment. We used Microwell-seq3 to profile chromatin accessibility in more than 200,000 single nuclei and the full-length transcriptome in ~50,000 nuclei from multiple adult mouse tissues. Compared with the existing polyadenylated transcript capture methods, integrative analysis of cell type-specific regulatory elements and total RNA expression uncovered comprehensive cell type heterogeneity in the brain. Gene regulatory networks based on chromatin accessibility profiling provided an improved cell type communication model. Finally, we demonstrated that Microwell-seq3 can identify malignant cells and their specific regulons in spontaneous lung tumors of aged mice. We envision a broad application of Microwell-seq3 in many areas of research.

Transposable Elements Are Differentially Expressed in MDS Stem Cells in a Disease-Stage-Specific Manner

Introduction Transposable elements (TEs) are widely spread across the human genome. Although TEs have been implicated in the pathogenesis of hematological malignancies, their expression in myelodysplastic syndromes (MDS) has not yet received extensive analysis. In the present study, we performed RNA sequence analysis of bone marrow stem and progenitor cells from patients with MDS and secondary acute myeloid leukemia (AML) and profiled differentially expressed TEs. Methods Bone marrow samples were obtained from each patient at the time of diagnosis. Normal bone marrow (NBM) samples from four healthy individuals were purchased from Lonza and processed in the same manner. After separation of CD34 + cells using magnetic beads, we purified lineage marker-negative (Lin -) CD34 + CD38 - stem cells and Lin - CD34 + CD38 + progenitor cells by cell sorting. The sorted cells were subjected to RNA-seq analysis using oligo-dT primers, which can capture poly-adenylated TE transcripts. Each read was aligned to the reference genome, then the annotation of the transcripts was performed using RepeatMasker, a program that screens DNA sequences for interspersed repeats and low-complexity DNA sequences (https://www.repeatmasker.org). Read counts for individual TEs were aggregated to the level of TE class, family, and subfamily according to hierarchies defined by the human RepeatMasker (repClass, repFamily, and repName, respectively). To generate raw counts of individual TE regions in RepeatMasker, the bedtools map function (version 2.27.1) was used with bed files of each sample. Normalization and significant expression differences were detected using DESeq2 (version 2.2.1). Differentially expressed TEs were defined using the cutoff of adjusted P values (q < 0.05). DESeq2 normalized counts were obtained from read counts by considering sequence depth, gene length, and RNA composition. Normalized counts were z-score scaled, and then subjected to uniform manifold approximation and projection (UMAP). Results Thirty-two patients were included in the present study. According to the 2016 revision to the World Health Organization classification (Arber DA, et al. Blood. 2016; 127: 2391-2405), 13 patients (40.6%) were diagnosed with MDS with multilineage dysplasia (MDS-MLD), 15 patients (46.9%) with MDS with excess blasts (MDS-EB), and 4 patients (12.5%) with AML with myelodysplasia-related changes (AML-MRC). We found that 26 and 17 repName TEs were significantly up-regulated in stem and progenitor cells compared with their NBM counterparts, and the majority of them belonged to LTRs, followed by DNA transposons. Since each LTR has multiple individual regions where similar sequences are scattered, LTR expression was further distinguished by the genomic locations of individual LTRs. Comparison of MDS-MLD, MDS-EB, and AML/MRC stem and progenitor cells to NBM counterparts revealed that more LTRs were differentially expressed in stem cells than in progenitor cells. Among them, many of the up-regulated LTRs were shared among different disease types (MDS-MLD, MDS-EB, and AML-MRC) in both stem and progenitor cells. AML-MRC had the largest number of differentially up- and down-regulated LTRs in stem cells. MDS-EB had the largest number of differentially up-regulated LTRs in progenitor cells, while only a small portion of LTRs were down-regulated in progenitor cells. LTRs at individual regions were differentially expressed in a more disease-specific manner, particularly in stem cells. Accordingly, UMAP plots that visualized the expression profiles of LTRs at individual regions showed that AML-MRC stem cells form a unique cluster, discriminating AML-MRC from MDS. To further understand the transcriptional reprogramming of LTRs during the disease progression from MDS to secondary AML, we analyzed the expression of differentially expressed LTRs at individual regions specific to AML-MRC by K-means clustering. They were subdivided into four clusters, of which Clusters 1, 3, and 4 showed gradual upregulation during disease progression, while Cluster 2 showed comparable upregulation at MDS and AML-MRC stages. Conclusions Our results suggest that specific LTRs behave in a disease-status and cell-type-specific manner in MDS and during leukemic transformation, providing fundamental information for understanding the pathogenesis of MDS.

Pan-Cancer Single-Nucleus Total RNA Sequencing Using snHH-Seq.

Tumor heterogeneity and its drivers impair tumor progression and cancer therapy. Single-cell RNA sequencing is used to investigate the heterogeneity of tumor ecosystems. However, most methods of scRNA-seq amplify the termini of polyadenylated transcripts, making it challenging to perform total RNA analysis and somatic mutation analysis.Therefore, a high-throughput and high-sensitivity method called snHH-seq is developed, which combines random primers and a preindex strategy in the droplet microfluidic platform. This innovative method allows for the detection of total RNA in single nuclei from clinically frozen samples. A robust pipeline to facilitate the analysis of full-length RNA-seq data is also established. snHH-seq is applied to more than 730000 single nuclei from 32 patients with various tumor types. The pan-cancer study enables it to comprehensively profile data on the tumor transcriptome, including expression levels, mutations, splicing patterns, clone dynamics, etc. New malignant cell subclusters and exploring their specific function across cancers are identified. Furthermore, the malignant status of epithelial cells is investigated among different cancer types with respect to mutation and splicing patterns. The ability to detect full-length RNA at the single-nucleus level provides a powerful tool for studying complex biological systems and has broad implications for understanding tumor pathology.

Transcript Isoform Diversity of Ampliconic Genes on the Y Chromosome of Great Apes.

Y chromosomal ampliconic genes (YAGs) are important for male fertility, as they encode proteins functioning in spermatogenesis. The variation in copy number and expression levels of these multicopy gene families has been studied in great apes; however, the diversity of splicing variants remains unexplored. Here, we deciphered the sequences of polyadenylated transcripts of all nine YAG families (BPY2, CDY, DAZ, HSFY, PRY, RBMY, TSPY, VCY, and XKRY) from testis samples of six great ape species (human, chimpanzee, bonobo, gorilla, Bornean orangutan, and Sumatran orangutan). To achieve this, we enriched YAG transcripts with capture probe hybridization and sequenced them with long (Pacific Biosciences) reads. Our analysis of this data set resulted in several findings. First, we observed evolutionarily conserved alternative splicing patterns for most YAG families except for BPY2 and PRY. Second, our results suggest that BPY2 transcripts and proteins originate from separate genomic regions in bonobo versus human, which is possibly facilitated by acquiring new promoters. Third, our analysis indicates that the PRY gene family, having the highest representation of noncoding transcripts, has been undergoing pseudogenization. Fourth, we have not detected signatures of selection in the five YAG families shared among great apes, even though we identified many species-specific protein-coding transcripts. Fifth, we predicted consensus disorder regions across most gene families and species, which could be used for future investigations of male infertility. Overall, our work illuminates the YAG isoform landscape and provides a genomic resource for future functional studies focusing on infertility phenotypes in humans and critically endangered great apes.

DNMT and HDAC inhibition induces immunogenic neoantigens from human endogenous retroviral element-derived transcripts

Immunotherapies targeting cancer-specific neoantigens have revolutionized the treatment of cancer patients. Recent evidence suggests that epigenetic therapies synergize with immunotherapies, mediated by the de-repression of endogenous retroviral element (ERV)-encoded promoters, and the initiation of transcription. Here, we use deep RNA sequencing from cancer cell lines treated with DNA methyltransferase inhibitor (DNMTi) and/or Histone deacetylase inhibitor (HDACi), to assemble a de novo transcriptome and identify several thousand ERV-derived, treatment-induced novel polyadenylated transcripts (TINPATs). Using immunopeptidomics, we demonstrate the human leukocyte antigen (HLA) presentation of 45 spectra-validated treatment-induced neopeptides (t-neopeptides) arising from TINPATs. We illustrate the potential of the identified t-neopeptides to elicit a T-cell response to effectively target cancer cells. We further verify the presence of t-neopeptides in AML patient samples after in vivo treatment with the DNMT inhibitor Decitabine. Our findings highlight the potential of ERV-derived neoantigens in epigenetic and immune therapies.

Single-cell lineage capture across genomic modalities with CellTag-multi reveals fate-specific gene regulatory changes.

Complex gene regulatory mechanisms underlie differentiation and reprogramming. Contemporary single-cell lineage-tracing (scLT) methods use expressed, heritable DNA barcodes to combine cell lineage readout with single-cell transcriptomics. However, reliance on transcriptional profiling limits adaptation to other single-cell assays. With CellTag-multi, we present an approach that enables direct capture of heritable random barcodes expressed as polyadenylated transcripts, in both single-cell RNA sequencing and single-cell Assay for Transposase Accessible Chromatin using sequencing assays, allowing for independent clonal tracking of transcriptional and epigenomic cell states. We validate CellTag-multi to characterize progenitor cell lineage priming during mouse hematopoiesis. Additionally, in direct reprogramming of fibroblasts to endoderm progenitors, we identify core regulatory programs underlying on-target and off-target fates. Furthermore, we reveal the transcription factor Zfp281 as a regulator of reprogramming outcome, biasing cells toward an off-target mesenchymal fate. Our results establish CellTag-multi as a lineage-tracing method compatible with multiple single-cell modalities and demonstrate its utility in revealing fate-specifying gene regulatory changes across diverse paradigms of differentiation and reprogramming.

NADcapPro and circNC: methods for accurate profiling of NAD and non-canonical RNA caps in eukaryotes

Accurate identification of NAD-capped RNAs is essential for delineating their generation and biological function. Previous transcriptome-wide methods used to classify NAD-capped RNAs in eukaryotes contain inherent limitations that have hindered the accurate identification of NAD caps from eukaryotic RNAs. In this study, we introduce two orthogonal methods to identify NAD-capped RNAs more precisely. The first, NADcapPro, uses copper-free click chemistry and the second is an intramolecular ligation-based RNA circularization, circNC. Together, these methods resolve the limitations of previous methods and allowed us to discover unforeseen features of NAD-capped RNAs in budding yeast. Contrary to previous reports, we find that 1) cellular NAD-RNAs can be full-length and polyadenylated transcripts, 2) transcription start sites for NAD-capped and canonical m7G-capped RNAs can be different, and 3) NAD caps can be added subsequent to transcription initiation. Moreover, we uncovered a dichotomy of NAD-RNAs in translation where they are detected with mitochondrial ribosomes but minimally on cytoplasmic ribosomes indicating their propensity to be translated in mitochondria.

The landscape of cryptic antisense transcription in human cancers reveals an oncogenic noncoding RNA in lung cancer.

Cryptic transcription initiation has been previously linked to activation of oncogenic transcripts. However, the prevalence and impact of cryptic antisense transcription from the opposite strand of protein-coding genes were mostly unknown in cancer. Applying a robust computational pipeline to publicly available transcriptome and epigenome datasets, we identified hundreds of previously unannotated cryptic antisense polyadenylated transcripts (CAPTs) that were enriched in tumor samples. We showed that the activation of cryptic antisense transcription was associated with increased chromatin accessibility and active histone marks. Accordingly, we found that many of the antisense transcripts were inducible by treatment of epigenetic drugs. Moreover, CRISPR-mediated epigenetic editing assays revealed that transcription of a noncoding RNA LRRK1-CAPT promoted LUSC cell proliferation, suggesting its oncogenic role. Our findings largely expand our understanding of cancer-associated transcription events, which may facilitate the development of novel strategies for cancer diagnosis and treatment.

PABPN1 prevents the nuclear export of an unspliced RNA with a constitutive transport element and controls human gene expression via intron retention.

Intron retention is a type of alternative splicing where one or more introns remain unspliced in a polyadenylated transcript. Although many viral systems are known to translate proteins from mRNAs with retained introns, restriction mechanisms generally prevent export and translation of incompletely spliced mRNAs. Here, we provide evidence that the human nuclear poly(A)-binding protein, PABPN1, functions in such restrictions. Using a reporter construct in which nuclear export of an incompletely spliced mRNA is enhanced by a viral constitutive transport element (CTE), we show that PABPN1 depletion results in a significant increase in export and translation from the unspliced CTE-containing transcript. Unexpectedly, we find that inactivation of poly(A)-tail exosome targeting by depletion of PAXT components had no effect on export and translation of the unspliced reporter mRNA, suggesting a mechanism largely independent of nuclear RNA decay. Interestingly, a PABPN1 mutant selectively defective in stimulating poly(A) polymerase elongation strongly enhanced the expression of the unspliced, but not of intronless, reporter transcripts. Analysis of RNA-seq data also revealed that PABPN1 controls the expression of many human genes via intron retention. Notably, PABPN1-dependent intron retention events mostly affected 3'-terminal introns and were insensitive to PAXT and NEXT deficiencies. Our findings thus disclose a role for PABPN1 in restricting nuclear export of intron-retained transcripts and reinforce the interdependence between terminal intron splicing, 3' end processing, and polyadenylation.

Human Satellite 1A analysis provides evidence of pericentromeric transcription

BackgroundPericentromeric regions of human chromosomes are composed of tandem-repeated and highly organized sequences named satellite DNAs. Human classical satellite DNAs are classified into three families named HSat1, HSat2, and HSat3, which have historically posed a challenge for the assembly of the human reference genome where they are misrepresented due to their repetitive nature. Although being known for a long time as the most AT-rich fraction of the human genome, classical satellite HSat1A has been disregarded in genomic and transcriptional studies, falling behind other human satellites in terms of functional knowledge. Here, we aim to characterize and provide an understanding on the biological relevance of HSat1A.ResultsThe path followed herein trails with HSat1A isolation and cloning, followed by in silico analysis. Monomer copy number and expression data was obtained in a wide variety of human cell lines, with greatly varying profiles in tumoral/non-tumoral samples. HSat1A was mapped in human chromosomes and applied in in situ transcriptional assays. Additionally, it was possible to observe the nuclear organization of HSat1A transcripts and further characterize them by 3′ RACE-Seq. Size-varying polyadenylated HSat1A transcripts were detected, which possibly accounts for the intricate regulation of alternative polyadenylation.ConclusionAs far as we know, this work pioneers HSat1A transcription studies. With the emergence of new human genome assemblies, acrocentric pericentromeres are becoming relevant characters in disease and other biological contexts. HSat1A sequences and associated noncoding RNAs will most certainly prove significant in the future of HSat research.

Principles of RNA recruitment to viral ribonucleoprotein condensates in a segmented dsRNA virus.

Rotaviruses transcribe 11 distinct RNAs that must be co-packaged prior to their replication to make an infectious virion. During infection, nontranslating rotavirus transcripts accumulate in cytoplasmic protein-RNA granules known as viroplasms that support segmented genome assembly and replication via a poorly understood mechanism. Here, we analysed the RV transcriptome by combining DNA-barcoded smFISH of rotavirus-infected cells. Rotavirus RNA stoichiometry in viroplasms appears to be distinct from the cytoplasmic transcript distribution, with the largest transcript being the most enriched in viroplasms, suggesting a selective RNA enrichment mechanism. While all 11 types of transcripts accumulate in viroplasms, their stoichiometry significantly varied between individual viroplasms. Accumulation of transcripts requires the presence of 3' untranslated terminal regions and viroplasmic localisation of the viral polymerase VP1, consistent with the observed lack of polyadenylated transcripts in viroplasms. Our observations reveal similarities between viroplasms and other cytoplasmic RNP granules and identify viroplasmic proteins as drivers of viral RNA assembly during viroplasm formation.

Single-cell transcriptomics and data analyses for prokaryotes-Past, present and future concepts.

Transcriptomics, or more specifically mRNA sequencing, is a powerful tool to study gene expression at the single-cell level (scRNA-seq) which enables new insights into a plethora of biological processes. While methods for single-cell RNA-seq in eukaryotes are well established, application to prokaryotes is still challenging. Reasons for that are rigid and diverse cell wall structures hampering lysis, the lack of polyadenylated transcripts impeding mRNA enrichment, and minute amounts of RNA requiring amplification steps before sequencing. Despite those obstacles, several promising scRNA-seq approaches for bacteria have been published recently, albeit difficulties in the experimental workflow and data processing and analysis remain. In particular, bias is often introduced by amplification which makes it difficult to distinguish between technical noise and biological variation. Future optimization of experimental procedures and data analysis algorithms are needed for the improvement of scRNA-seq but also to aid in the emergence of prokaryotic single-cell multi-omics. to help address 21st century challenges in the biotechnology and health sector.

Spatial mapping of the total transcriptome by in situ polyadenylation

Spatial transcriptomics reveals the spatial context of gene expression, but current methods are limited to assaying polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNA enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based spatial transcriptomics to the total transcriptome. We demonstrate that our spatial total RNA-sequencing (STRS) approach captures coding RNAs, noncoding RNAs and viral RNAs. We apply STRS to study skeletal muscle regeneration and viral-induced myocarditis. Our analyses reveal the spatial patterns of noncoding RNA expression with near-cellular resolution, identify spatially defined expression of noncoding transcripts in skeletal muscle regeneration and highlight host transcriptional responses associated with local viral RNA abundance. STRS requires adding only one step to the widely used Visium spatial total RNA-sequencing protocol from 10x Genomics, and thus could be easily adopted to enable new insights into spatial gene regulation and biology.

Alternative cleavage and polyadenylation generates downstream uncapped RNA isoforms with translation potential

The use of alternative promoters, splicing, and cleavage and polyadenylation (APA) generates mRNA isoforms that expand the diversity and complexity of the transcriptome. Here, we uncovered thousands of previously undescribed 5' uncapped and polyadenylated transcripts (5' UPTs). We show that these transcripts resist exonucleases due to a highly structured RNA and N6-methyladenosine modification at their 5' termini. 5' UPTs appear downstream of APA sites within their host genes and are induced upon APA activation. Strong enrichment in polysomal RNA fractions indicates 5' UPT translational potential. Indeed, APA promotes downstream translation initiation, non-canonical protein output, and consistent changes to peptide presentation at the cell surface. Lastly, we demonstrate the biological importance of 5' UPTs using Bcl2, a prominent anti-apoptotic gene whose entire coding sequence is a 5' UPT generated from 5' UTR-embedded APA sites. Thus, APA is not only accountable for terminating transcripts, but also for generating downstream uncapped RNAs with translation potential and biological impact.