Poly(A) tale: From A to A; RNA polyadenylation in prokaryotes and eukaryotes.

Most eukaryotic mRNAs and different non-coding RNAs undergo a form of 3' end processing known as polyadenylation. Polyadenylation machinery is present in almost all organisms except few species. In bacteria, the machinery has evolved from PNPase, which adds heteropolymeric tails, to a poly(A)-specific polymerase. Differently, a complex machinery for accurate polyadenylation and several non-canonical poly(A) polymerases are developed in eukaryotes. The role of poly(A) tail has also evolved from serving as a degradative signal to a stabilizing modification that also regulates translation. In this review, we discuss poly(A) tail emergence in prokaryotes and its development into a stable, yet dynamic feature at the 3' end of mRNAs in eukaryotes. We also describe how appearance of novel poly(A) polymerases gives cells flexibility to shape poly(A) tail. We explain how poly(A) tail dynamics help regulate cognate RNA metabolism in a context-dependent manner, such as during oocyte maturation. Finally, we describe specific mRNAs in metazoans that bear stem-loops instead of poly(A) tails. We conclude with how recent discoveries about poly(A) tail can be applied to mRNA technology. This article is categorized under: RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Processing > 3' End Processing RNA Turnover and Surveillance > Regulation of RNA Stability.

Time-resolved Small-RNA Sequencing Identifies MicroRNAs Critical for Formation of Embryonic Stem Cells from the Inner Cell Mass of Mouse Embryos.

Cells of the inner cell mass (ICM) acquire a unique ability for unlimited self-renewal during transition into embryonic stem cells (ESCs) in vitro, while preserving their natural multi-lineage differentiation potential. Several different pathways have been identified to play roles in ESC formation but the function of non-coding RNAs in this process is poorly understood. Here, we describe several microRNAs (miRNAs) that are crucial for efficient generation of mouse ESCs from ICMs. Using small-RNA sequencing, we characterize dynamic changes in miRNA expression profiles during outgrowth of ICMs in a high-resolution, time-course dependent manner. We report several waves of miRNA transcription during ESC formation, to which miRNAs from the imprinted Dlk1-Dio3 locus contribute extensively. In silico analyses followed by functional investigations reveal that Dlk1-Dio3 locus-embedded miRNAs (miR-541-5p, miR-410-3p, and miR-381-3p), miR-183-5p, and miR-302b-3p promote, while miR-212-5p and let-7d-3p inhibit ESC formation. Collectively, these findings offer new mechanistic insights into the role of miRNAs during ESC derivation.

SARS-CoV-2 mRNA-vaccine candidate; COReNAPCIN®, induces robust humoral and cellular immunity in mice and non-human primates.

At the forefront of biopharmaceutical industry, the messenger RNA (mRNA) technology offers a flexible and scalable platform to address the urgent need for world-wide immunization in pandemic situations. This strategic powerful platform has recently been used to immunize millions of people proving both of safety and highest level of clinical efficacy against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we provide preclinical report of COReNAPCIN®; a vaccine candidate against SARS-CoV-2 infection. COReNAPCIN® is a nucleoside modified mRNA-based vaccine formulated in lipid nanoparticles (LNPs) for encoding the full-length prefusion stabilized SARS-CoV-2 spike glycoprotein on the cell surface. Vaccination of C57BL/6 and BALB/c mice and rhesus macaque with COReNAPCIN® induced strong humoral responses with high titers of virus-binding and neutralizing antibodies. Upon vaccination, a robust SARS-CoV-2 specific cellular immunity was also observed in both mice and non-human primate models. Additionally, vaccination protected rhesus macaques from symptomatic SARS-CoV-2 infection and pathological damage to the lung upon challenging the animals with high viral loads of up to 2 × 108 live viral particles. Overall, our data provide supporting evidence for COReNAPCIN® as a potent vaccine candidate against SARS-CoV-2 infection for clinical studies.

Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution.

The breadth and importance of RNA modifications are growing rapidly as modified ribonucleotides can impact the sequence, structure, function, stability, and fate of RNAs and their interactions with other molecules. Therefore, knowing cellular RNA modifications at single-base resolution could provide important information regarding cell status and fate. A current major limitation is the lack of methods that allow the reproducible profiling of multiple modifications simultaneously, transcriptome-wide and at single-base resolution. Here we developed RBS-Seq, a modification of RNA bisulfite sequencing that enables the sensitive and simultaneous detection of m5C, Ψ, and m1A at single-base resolution transcriptome-wide. With RBS-Seq, m5C and m1A are accurately detected based on known signature base mismatches and are detected here simultaneously along with Ψ sites that show a 1-2 base deletion. Structural analyses revealed the mechanism underlying the deletion signature, which involves Ψ-monobisulfite adduction, heat-induced ribose ring opening, and Mg2+-assisted reorientation, causing base-skipping during cDNA synthesis. Detection of each of these modifications through a unique chemistry allows high-precision mapping of all three modifications within the same RNA molecule, enabling covariation studies. Application of RBS-Seq on HeLa RNA revealed almost all known m5C, m1A, and ψ sites in tRNAs and rRNAs and provided hundreds of new m5C and Ψ sites in noncoding RNAs and mRNAs. However, our results diverge greatly from earlier work, suggesting ∼10-fold fewer m5C sites in noncoding and coding RNAs and the absence of substantial m1A in mRNAs. Taken together, the approaches and refined datasets in this work will greatly enable future epitranscriptome studies.

Active BRAF-V600E is the key player in generation of a sessile serrated polyp-specific DNA methylation profile.

BACKGROUND: Sessile serrated polyps (SSPs) have emerged as important precursors for a large number of sporadic colorectal cancers. They are difficult to detect during colonoscopy due to their flat shape and the excessive amounts of secreted mucin that cover the polyps. The underlying genetic and epigenetic basis for the emergence of SSPs is largely unknown with existing genetic studies confined to a limited number of oncogenes and tumor suppressors. A full characterization of the genetic and epigenetic landscape of SSPs would provide insight into their origin and potentially offer new biomarkers useful for detection of SSPs in stool samples. METHODS: We used a combination of genome-wide mutation detection, exome sequencing and DNA methylation profiling (via methyl-array and whole-genome bisulfite sequencing) to analyze multiple samples of sessile serrated polyps and compared these to familial adenomatous polyps. RESULTS: Our analysis revealed BRAF-V600E as the sole recurring somatic mutation in SSPs with no additional major genetic mutations detected. The occurrence of BRAF-V600E was coincident with a unique DNA methylation pattern revealing a set of DNA methylation markers showing significant (~3 to 30 fold) increase in their methylation levels, exclusively in SSP samples. These methylation patterns effectively distinguished sessile serrated polys from adenomatous polyps and did so more effectively than parallel gene expression profiles. CONCLUSIONS: This study provides an important example of a single oncogenic mutation leading to reproducible global DNA methylation changes. These methylated markers are specific to SSPs and could be of important clinical relevance for the early diagnosis of SSPs using non-invasive approaches such as fecal DNA testing.

Experimental Approaches for Target Profiling of RNA Cytosine Methyltransferases.

RNA cytosine methyltransferases (m(5)C-RMTs) constitute an important class of RNA-modifying enzymes, methylating specific cytosines within particular RNA targets in both coding and noncoding RNAs. Almost all organisms express at least one m(5)C-RMT, and vertebrates often express different types or variants of m(5)C-RMTs in different cell types. Deletion or mutation of particular m(5)C-RMTs is connected to severe pathological manifestations ranging from developmental defects to infertility and mental retardation. Some m(5)C-RMTs show spatiotemporal patterns of expression and activity requiring careful experimental design for their analysis in order to capture their context-dependent targets. An essential step for understanding the functions of both the enzymes and the modified cytosines is defining the one-to-one connection between particular m(5)C-RMTs and their target cytosines. Recent technological and methodological advances have provided researchers with new tools to comprehensively explore RNA cytosine methylation and methyltransferases. Here, we describe three complementary approaches applicable for both discovery and validation of candidate target sites of specific m(5)C-RMTs.

Pluripotency transcription factor Oct4 mediates stepwise nucleosome demethylation and depletion.

The mechanisms whereby the crucial pluripotency transcription factor Oct4 regulates target gene expression are incompletely understood. Using an assay system based on partially differentiated embryonic stem cells, we show that Oct4 opposes the accumulation of local H3K9me2 and subsequent Dnmt3a-mediated DNA methylation. Upon binding DNA, Oct4 recruits the histone lysine demethylase Jmjd1c. Chromatin immunoprecipitation (ChIP) time course experiments identify a stepwise Oct4 mechanism involving Jmjd1c recruitment and H3K9me2 demethylation, transient FACT (facilitates chromatin transactions) complex recruitment, and nucleosome depletion. Genome-wide and targeted ChIP confirms binding of newly synthesized Oct4, together with Jmjd1c and FACT, to the Pou5f1 enhancer and a small number of other Oct4 targets, including the Nanog promoter. Histone demethylation is required for both FACT recruitment and H3 depletion. Jmjd1c is required to induce endogenous Oct4 expression and fully reprogram fibroblasts to pluripotency, indicating that the assay system identifies functional Oct4 cofactors. These findings indicate that Oct4 sequentially recruits activities that catalyze histone demethylation and depletion.

Transcriptome-wide target profiling of RNA cytosine methyltransferases using the mechanism-based enrichment procedure Aza-IP.

Cytosine methylation within RNA is common, but its full scope and functions are poorly understood, as the RNA targets of most mammalian cytosine RNA methyltransferases (m(5)C-RMTs) remain uncharacterized. To enable their characterization, we developed a mechanism-based method for transcriptome-wide m(5)C-RMT target profiling. All characterized mammalian m(5)C-RMTs form a reversible covalent intermediate with their cytosine substrate-a covalent linkage that is trapped when conducted on the cytosine analog 5-azacytidine (5-aza-C). We used this property to develop Aza-immunoprecipitation (Aza-IP), a methodology to form stable m(5)C-RMT-RNA linkages in cell culture, followed by IP and high-throughput sequencing, to identify direct RNA substrates of m(5)C-RMTs. Remarkably, a cytosine-to-guanine (C→G) transversion occurs specifically at target cytosines, allowing the simultaneous identification of the precise target cytosine within each RNA. Thus, Aza-IP reports only direct RNA substrates and the C→G transversion provides an important criterion for target cytosine identification, which is not available in alternative approaches. Here we present a step-by-step protocol for Aza-IP and downstream analysis, designed to reveal identification of substrate RNAs and precise cytosine targets of m(5)C-RMTs. The entire protocol takes 40-50 d to complete.

Identification of direct targets and modified bases of RNA cytosine methyltransferases.

The extent and biological impact of RNA cytosine methylation are poorly understood, in part owing to limitations of current techniques for determining the targets of RNA methyltransferases. Here we describe 5-azacytidine-mediated RNA immunoprecipitation (Aza-IP), a technique that exploits the covalent bond formed between an RNA methyltransferase and the cytidine analog 5-azacytidine to recover RNA targets by immunoprecipitation. Targets are subsequently identified by high-throughput sequencing. When applied in a human cell line to the RNA methyltransferases DNMT2 and NSUN2, Aza-IP enabled >200-fold enrichment of tRNAs that are known targets of the enzymes. In addition, it revealed many tRNA and noncoding RNA targets not previously associated with NSUN2. Notably, we observed a high frequency of C→G transversions at the cytosine residues targeted by both enzymes, allowing identification of the specific methylated cytosine(s) in target RNAs. Given the mechanistic similarity of RNA cytosine methyltransferases, Aza-IP may be generally applicable for target identification.