Direct RNA sequencing in plants: Practical applications and future perspectives.

The transcriptome serves as a bridge that links genomic variation to phenotypic diversity. A vast number of studies using next-generation RNA sequencing (RNA-seq) over the last 2 decades have emphasized the essential roles of the plant transcriptome in response to developmental and environmental conditions, providing numerous insights into the dynamic changes, evolutionary traces, and elaborate regulation of the plant transcriptome. With substantial improvement in accuracy and throughput, direct RNA sequencing (DRS) has emerged as a new and powerful sequencing platform for precise detection of native and full-length transcripts, overcoming many limitations such as read length and PCR bias that are inherent to short-read RNA-seq. Here, we review recent advances in dissecting the complexity and diversity of plant transcriptomes using DRS as the main technological approach, covering many aspects of RNA metabolism, including novel isoforms, poly(A) tails, and RNA modification, and we propose a comprehensive workflow for processing of plant DRS data. Many challenges to the application of DRS in plants, such as the need for machine learning tools tailored to plant transcriptomes, remain to be overcome, and together we outline future biological questions that can be addressed by DRS, such as allele-specific RNA modification. This technology provides convenient support on which the connection of distinct RNA features is tightly built, sustainably refining our understanding of the biological functions of the plant transcriptome.

Evolution and post-transcriptional regulation insights of m6A writers, erasers, and readers in plant epitranscriptome.

As a dynamic and reversible post-transcriptional marker, N6-methyladenosine (m6A) plays an important role in the regulation of biological functions, which are mediated by m6A pathway components including writers (MT-A70, FIP37, VIR and HAKAI family), erasers (ALKBH family) and readers (YTH family). There is an urgent need for a comprehensive analysis of m6A pathway components across species at evolutionary levels. In this study, we identified 4062 m6A pathway components from 154 plant species including green algae, utilizing large-scale phylogenetic to explore their origin and evolution. We discovered that the copy number of writers was conserved among different plant lineages, with notable expansions in the ALKBH and YTH families. Synteny network analysis revealed conserved genomic contexts and lineage-specific transpositions. Furthermore, we used Direct RNA Sequencing (DRS) to reveal the Poly(A) length (PAL) and m6A ratio profiles in six angiosperms species, with a particular focus on the m6A pathway components. The ECT1/2-Poeaece4 sub-branches (YTH family) with unique genomic contexts exhibited significantly higher expression level than genes of other ECT1/2 poeaece sub-branches (ECT1/2-Poeaece1-3), accompanied by lower m6A modification and PAL. Besides, conserved m6A sites distributed in CDS and 3'UTR were detected in the ECT1/2-Poaceae4, and the dual-luciferase assay further demonstrated that these conserved m6A sites in the 3'UTR negatively regulated the expression of Firefly luciferase (LUC) gene. Finally, we developed transcription factor regulatory networks for m6A pathway components, using yeast one-hybrid assay demonstrated that PheBPC1 could interact with the PheECT1/2-5 promoter. Overall, this study presents a comprehensive evolutionary and functional analysis of m6A pathway components and their modifications in plants, providing a valuable resource for future functional analysis in this field.

Prognostic insights, immune infiltration, and therapeutic response: Cytoplasmic poly(A) tail regulators in hepatocellular carcinoma.

The presence of a poly(A) tail is indispensable for the post-transcriptional regulation of gene expression in cancer. This dynamic and modifiable feature of transcripts is under the control of various nuclear and cytoplasmic proteins. This study aimed to develop a novel cytoplasmic poly(A)-related signature for predicting prognosis, clinical attributes, tumor immune microenvironment (TIME), and treatment response in hepatocellular carcinoma (HCC). Utilizing RNA sequencing (RNA-seq) data from The Cancer Genome Atlas (TCGA), non-negative matrix factorization (NMF), and principal-component analysis (PCA) were employed to categorize HCC patients into three clusters, thus demonstrating the pivotal prognostic role of cytoplasmic poly(A) tail regulators. Furthermore, machine learning algorithms such as least absolute shrinkage and selection operator (LASSO), survival analysis, and Cox proportional hazards modeling were able to distinguish distinct cytoplasmic poly(A) subtypes. As a result, a 5-gene signature derived from TCGA was developed and validated using International Cancer Genome Consortium (ICGC) HCC datasets. This novel classification based on cytoplasmic poly(A) regulators has the potential to improve prognostic predictions and provide guidance for chemotherapy, immunotherapy, and transarterial chemoembolization (TACE) in HCC.

Base preference for inosine 3'-riboendonuclease activity of human endonuclease V: implications for cleavage of poly-A tails containing inosine.

Deamination of bases is a form of DNA damage that occurs spontaneously via the hydrolysis and nitrosation of living cells, generating hypoxanthine from adenine. E. coli endonuclease V (eEndoV) cleaves hypoxanthine-containing double-stranded DNA, whereas human endonuclease V (hEndoV) cleaves hypoxanthine-containing RNA; however, hEndoV in vivo function remains unclear. To date, hEndoV has only been examined using hypoxanthine, because it binds closely to the base located at the cleavage site. Here, we examined whether hEndoV cleaves other lesions (e.g., AP site, 6-methyladenine, xanthine) to reveal its function and whether 2'-nucleoside modification affects its cleavage activity. We observed that hEndoV is hypoxanthine-specific; its activity was the highest with 2'-OH modification in ribose. The cleavage activity of hEndoV was compared based on its base sequence. We observed that it has specificity for adenine located on the 3'-end of hypoxanthine at the cleavage site, both before and after cleavage. These data suggest that hEndoV recognizes and cleaves the inosine generated on the poly A tail to maintain RNA quality. Our results provide mechanistic insight into the role of hEndoV in vivo.

Beyond simple tails: poly(A) tail-mediated RNA epigenetic regulation.

The poly(A) tail is an essential structural component of mRNA required for the latter's stability and translation. Recent technologies have enabled transcriptome-wide profiling of the length and composition of poly(A) tails, shedding light on their overlooked regulatory capacities. Notably, poly(A) tails contain not only adenine but also uracil, cytosine, and guanine residues. These findings strongly suggest that poly(A) tails could encode a wealth of regulatory information, similar to known reversible RNA chemical modifications. This review aims to succinctly summarize our current knowledge on the composition, dynamics, and regulatory functions of RNA poly(A) tails. Given their capacity to carry rich regulatory information beyond the genetic code, we propose the concept of 'poly(A) tail epigenetic information' as a new layer of RNA epigenetic regulation.

Enhanced binding of guanylated poly(A) RNA by the LaM domain of LARP1.

La-related proteins (LARPs) are a family of RNA-binding proteins that share a conserved La motif (LaM) domain. LARP1 plays a role in regulating ribosomal protein synthesis and stabilizing mRNAs and has a unique structure without an RNA binding RRM domain adjoining the LaM domain. In this study, we investigated the physical basis for LARP1 specificity for poly(A) sequences and observed an unexpected bias for sequences with single guanines. Multiple guanine substitutions did not increase the affinity, demonstrating preferential recognition of singly guanylated sequences. We also observed that the cyclic di-nucleotides in the cCAS/STING pathway, cyclic-di-GMP and 3',3'-cGAMP, bound with sub-micromolar affinity. Isothermal titration measurements were complemented by high-resolution crystal structures of the LARP1 LaM with six different RNA ligands, including two stereoisomers of a phosphorothioate linkage. The selectivity for singly substituted poly(A) sequences suggests LARP1 may play a role in the stabilizing effect of poly(A) tail guanylation. [Figure: see text].

Decoupled degradation and translation enables noise modulation by poly(A) tails.

Poly(A) tails are crucial for mRNA translation and degradation, but the exact relationship between tail length and mRNA kinetics remains unclear. Here, we employ a small library of identical mRNAs that differ only in their poly(A)-tail length to examine their behavior in human embryonic kidney cells. We find that tail length strongly correlates with mRNA degradation rates but is decoupled from translation. Interestingly, an optimal tail length of ∼100 nt displays the highest translation rate, which is identical to the average endogenous tail length measured by nanopore sequencing. Furthermore, poly(A)-tail length variability-a feature of endogenous mRNAs-impacts translation efficiency but not mRNA degradation rates. Stochastic modeling combined with single-cell tracking reveals that poly(A) tails provide cells with an independent handle to tune gene expression fluctuations by decoupling mRNA degradation and translation. Together, this work contributes to the basic understanding of gene expression regulation and has potential applications in nucleic acid therapeutics.

Quality Control of mRNA Vaccines by Synthetic Ribonucleases: Analysis of the Poly-A-Tail.

The effectivity and safety of mRNA vaccines critically depends on the presence of correct 5' caps and poly-A tails. Due to the high molecular mass of full-size mRNAs, however, the direct analysis by mass spectrometry is hardly possible. Here we describe the use of synthetic ribonucleases to cleave off 5' and 3' terminal fragments which can be further analyzed by HPLC or by LC-MS. Compared to existing methods (e. g. RNase H), the new approach uses robust catalysts, is free of sequence limitations, avoids metal ions and combines fast sample preparation with high precision of the cut.

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.

Evaluation of size-exclusion chromatography, multi-angle light scattering detection and mass photometry for the characterization of mRNA.

The recent approval of messenger ribonucleic acid (mRNA) as vaccine to combat the COVID-19 pandemic has been a scientific turning point. Today, the applicability of mRNA is being demonstrated beyond infectious diseases, for example in cancer immunotherapy, protein replacement therapy and gene editing. mRNA is produced by in vitro transcription (IVT) from a linear DNA template and modified at the 3' and 5' ends to improve translational efficiency and stability. Co-existing impurities such as RNA fragments and double-stranded RNA (dsRNA), amongst others, can drastically impact mRNA quality and efficacy. In this study, size-exclusion chromatography (SEC) is evaluated for the characterization of IVT-mRNA. The effect of mobile phase composition (ionic strength and organic modifier), pH, column temperature and pore size (300 Å, 1000 Å, and 2000 Å) on the separation performance and structural integrity of IVT-mRNA varying in size is described. Non-replicating, self-amplifying (saRNA), temperature degraded, and ribonuclease (RNase) digested mRNA, the latter to characterize the 3' poly(A) tail, were included in the study. Beyond ultraviolet (UV) detection, refractive index (RI) and multi-angle light scattering (MALS) detection were implemented to accurately determine molecular weight (MW) of mRNA. Finally, mass photometry is introduced as a complementary methodology to study mRNA under native conditions.

Critical view on oligo(dT)-based RNA-seq: bias arising, modeling, and mitigating.

The precise biological interpretation of oligo(dT)-based RNA sequencing (RNA-seq) datasets, particularly in single-cell RNA-seq (scRNA-seq), is invaluable for understanding complex biological systems. However, the presence of biases can lead to misleading results in downstream analysis. This study has now identified two additional biases that are not accounted for in established bias models: poly(A)-tail length bias and fixed-position GC-content bias. These biases have a significant negative impact on the overall quality of oligo(dT)-based RNA-seq data. To address these biases, we have developed a universal bias-mitigating method based on the lower-affinity binding of short and nonanchored oligo(dT) primers to poly(A) tails. This method significantly reduces poly(A) length bias and completely eliminates fixed-position GC bias. Furthermore, the use of short oligo(dT) with impartial binding behavior toward the diverse poly(A) tails renders RNA-seq with more reliable measurements. The findings of this study are particularly beneficial for scRNA-seq datasets, where accurate benchmarking is critical.

Decoding the Heterogeneity and Specialized Function of Translation Machinery Through Ribosome Profiling in Yeast Mutants of Initiation Factors.

The nuanced heterogeneity and specialized functions of translation machinery are increasingly recognized as crucial for precise translational regulation. Here, high-throughput ribosomal profiling (ribo-seq) is used to analyze the specialized roles of eukaryotic initiation factors (eIFs) in the budding yeast. By examining changes in ribosomal distribution across the genome resulting from knockouts of eIF4A, eIF4B, eIF4G1, CAF20, or EAP1, or knockdowns of eIF1, eIF1A, eIF4E, or PAB1, two distinct initiation-factor groups, the "looping" and "scanning" groups are discerned, based on similarities in the ribosomal landscapes their perturbation induced. The study delves into the cis-regulatory sequence features of genes influenced predominantly by each group, revealing that genes more dependent on the looping-group factors generally have shorter transcripts and poly(A) tails. In contrast, genes more dependent on the scanning-group factors often possess upstream open reading frames and exhibit a higher GC content in their 5' untranslated regions. From the ribosomal RNA fragments identified in the ribo-seq data, ribosomal heterogeneity associated with perturbation of specific initiation factors is further identified, suggesting their potential roles in regulating ribosomal components. Collectively, the study illuminates the complexity of translational regulation driven by heterogeneity and specialized functions of translation machinery, presenting potential approaches for targeted gene translation manipulation.

Comprehensive gene profiling of the metabolic landscape of humanized livers in mice.

BACKGROUND & AIMS: The human liver transcriptome is complex and highly dynamic, e.g. one gene may produce multiple distinct transcripts, each with distinct posttranscriptional modifications. Direct knowledge of transcriptome dynamics, however, is largely obscured by the inaccessibility of the human liver to treatments and the insufficient annotation of the human liver transcriptome at transcript and RNA modification levels. METHODS: We generated mice that carry humanized livers of identical genetic background and subjected them to representative metabolic treatments. We then analyzed the humanized livers with nanopore single-molecule direct RNA sequencing to determine the expression level, m6A modification and poly(A) tail length of all RNA transcript isoforms. Our system allows for the de novo annotation of human liver transcriptomes to reflect metabolic responses and for the study of transcriptome dynamics in parallel. RESULTS: Our analysis uncovered a vast number of novel genes and transcripts. Our transcript-level analysis of human liver transcriptomes also identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional short-read RNA sequencing. We revealed for the first time the dynamic changes in m6A and poly(A) tail length of human liver transcripts, many of which are transcribed from key metabolic genes. Furthermore, we performed comparative analyses of gene regulation between humans and mice, and between two individuals using the liver-specific humanized mice, revealing that transcriptome dynamics are highly species- and genetic background-dependent. CONCLUSION: Our work revealed a complex metabolic response landscape of the human liver transcriptome and provides a novel resource to understand transcriptome dynamics of the human liver in response to physiologically relevant metabolic stimuli (https://caolab.shinyapps.io/human_hepatocyte_landscape/). IMPACT AND IMPLICATIONS: Direct knowledge of the human liver transcriptome is currently very limited, hindering the overall understanding of human liver pathophysiology. We combined a liver-specific humanized mouse model and long-read direct RNA sequencing technology to establish a de novo annotation of the human liver transcriptome and identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional technologies. The extensive regulatory information on human genes we provided could enable basic scientists to infer the pathological relevance of their genes of interest and physician scientists to better pinpoint the changes in metabolic networks underlying a specific pathophysiology.

The CARBON CATABOLITE REPRESSION 4A-mediated RNA deadenylation pathway acts on the transposon RNAs that are not regulated by small RNAs.

Transposable elements (TEs) are mobile genetic elements that can impair the host genome stability and integrity. It has been well documented that activated transposons in plants are suppressed by small interfering (si) RNAs. However, transposon repression by the cytoplasmic RNA surveillance system is unknown. Here, we show that mRNA deadenylation is critical for controlling transposons in Arabidopsis. Trimming of poly(A) tail is a rate-limiting step that precedes the RNA decay and is primarily mediated by the CARBON CATABOLITE REPRESSION 4 (CCR4)-NEGATIVE ON TATA-LESS (NOT) complex. We found that the loss of CCR4a leads to strong derepression and mobilization of TEs in Arabidopsis. Intriguingly, CCR4a regulates a largely distinct set of TEs from those controlled by RNA-dependent RNA Polymerase 6 (RDR6), a key enzyme that produces cytoplasmic siRNAs. This indicates that the cytoplasmic RNA quality control mechanism targets the TEs that are poorly recognized by the previously well-characterized RDR6-mediated pathway, and thereby augments the host genome stability. Our study suggests a hitherto unknown mechanism for transposon repression mediated by RNA deadenylation and unveils a complex nature of the host's strategy to maintain the genome integrity.

A direct interaction between CPF and RNA Pol II links RNA 3' end processing to transcription.

Transcription termination by RNA polymerase II (RNA Pol II) is linked to RNA 3' end processing by the cleavage and polyadenylation factor (CPF or CPSF). CPF contains endonuclease, poly(A) polymerase, and protein phosphatase activities, which cleave and polyadenylate pre-mRNAs and dephosphorylate RNA Pol II to control transcription. Exactly how the RNA 3' end processing machinery is coupled to transcription remains unclear. Here, we combine in vitro reconstitution, structural studies, and genome-wide analyses to show that yeast CPF physically and functionally interacts with RNA Pol II. Surprisingly, CPF-mediated dephosphorylation promotes the formation of an RNA Pol II stalk-to-stalk homodimer in vitro. This dimer is compatible with transcription but not with the binding of transcription elongation factors. Disruption of the dimerization interface in cells causes transcription defects, including altered RNA Pol II abundance on protein-coding genes, tRNA genes, and intergenic regions. We hypothesize that RNA Pol II dimerization may provide a mechanistic basis for the allosteric model of transcription termination.

CLT-seq as a universal homopolymer-sequencing concept reveals poly(A)-tail-tuned ncRNA regulation.

Dynamic tuning of the poly(A) tail is a crucial mechanism for controlling translation and stability of eukaryotic mRNA. Achieving a comprehensive understanding of how this regulation occurs requires unbiased abundance quantification of poly(A)-tail transcripts and simple poly(A)-length measurement using high-throughput sequencing platforms. Current methods have limitations due to complicated setups and elaborate library preparation plans. To address this, we introduce central limit theorem (CLT)-managed RNA-seq (CLT-seq), a simple and straightforward homopolymer-sequencing method. In CLT-seq, an anchor-free oligo(dT) primer rapidly binds to and unbinds from anywhere along the poly(A) tail string, leading to position-directed reverse transcription with equal probability. The CLT mechanism enables the synthesized poly(T) lengths, which correspond to the templated segment of the poly(A) tail, to distribute normally. Based on a well-fitted pseudogaussian-derived poly(A)-poly(T) conversion model, the actual poly(A)-tail profile is reconstructed from the acquired poly(T)-length profile through matrix operations. CLT-seq follows a simple procedure without requiring RNA-related pre-treatment, enrichment or selection, and the CLT-shortened poly(T) stretches are more compatible with existing sequencing platforms. This proof-of-concept approach facilitates direct homopolymer base-calling and features unbiased RNA-seq. Therefore, CLT-seq provides unbiased, robust and cost-efficient transcriptome-wide poly(A)-tail profiling. We demonstrate that CLT-seq on the most common Illumina platform delivers reliable poly(A)-tail profiling at a transcriptome-wide scale in human cellular contexts. We find that the poly(A)-tail-tuned ncRNA regulation undergoes a dynamic, complex process similar to mRNA regulation. Overall, CLT-seq offers a simplified, effective and economical approach to investigate poly(A)-tail regulation, with potential implications for understanding gene expression and identifying therapeutic targets.

A single-nucleotide resolution capillary gel electrophoresis workflow for poly(A) tail characterization in the development of mRNA therapeutics and vaccines.

The 3' poly(A) tail is an important component of messenger RNA (mRNA). The length of the poly(A) tail has direct impact on the stability and translation efficiency of the mRNA molecule and is therefore considered to be a critical quality attribute (CQA) of mRNA-based therapeutics and vaccines. Various analytical methods have been developed to monitor this CQA. Methods like ion-pair reversed-phase liquid chromatography (IPRP-LC) can be used to quantify the percentage of mRNA with poly(A) tail but fail to provide further information on the actual length of poly(A). High-resolution methods such as liquid chromatography coupled with mass spectrometry (LC-MS) or next generation sequencing (NGS) can separate poly(A) tail length by one nucleotide (n/n + 1 resolution) but are complicated to implement for release testing of manufactured mRNA. In this study, a workflow utilizing capillary gel electrophoresis (CGE) for characterizing the poly(A) tail length of mRNA was developed. The CGE method demonstrated resolution comparable with the LC-MS method. With UV detection and the addition of poly(A) length markers, this method can provide poly(A) tail length information and can also provide quantitation of each poly(A) length, making it a suitable release method to monitor the CQA of poly(A) tail length.

The mRNA Vaccine Revolution: COVID-19 Has Launched the Future of Vaccinology.

During the COVID-19 pandemic, mRNA (mRNA) vaccines emerged as leading vaccine candidates in a record time. Nonreplicating mRNA (NRM) and self-amplifying mRNA (SAM) technologies have been developed into high-performing and clinically viable vaccines against a range of infectious agents, notably SARS-CoV-2. mRNA vaccines demonstrate efficient in vivo delivery, long-lasting stability, and nonexistent risk of infection. The stability and translational efficiency of in vitro transcription (IVT)-mRNA can be further increased by modulating its structural elements. In this review, we present a comprehensive overview of the recent advances, key applications, and future challenges in the field of mRNA-based vaccinology.

Functional viromic screens uncover regulatory RNA elements.

The number of sequenced viral genomes has surged recently, presenting an opportunity to understand viral diversity and uncover unknown regulatory mechanisms. Here, we conducted a screening of 30,367 viral segments from 143 species representing 96 genera and 37 families. Using a library of viral segments in 3' UTR, we identified hundreds of elements impacting RNA abundance, translation, and nucleocytoplasmic distribution. To illustrate the power of this approach, we investigated K5, an element conserved in kobuviruses, and found its potent ability to enhance mRNA stability and translation in various contexts, including adeno-associated viral vectors and synthetic mRNAs. Moreover, we identified a previously uncharacterized protein, ZCCHC2, as a critical host factor for K5. ZCCHC2 recruits the terminal nucleotidyl transferase TENT4 to elongate poly(A) tails with mixed sequences, delaying deadenylation. This study provides a unique resource for virus and RNA research and highlights the potential of the virosphere for biological discoveries.

Inhibition of DNA and RNA methylation disturbs root development of moso bamboo.

DNA methylation (5mC) and N6-methyladenosine (m6A) are two important epigenetics regulators, which have a profound impact on plant growth development. Phyllostachys edulis (P. edulis) is one of the fastest spreading plants due to its well-developed root system. However, the association between 5mC and m6A has seldom been reported in P. edulis. In particular, the connection between m6A and several post-transcriptional regulators remains uncharacterized in P. edulis. Here, our morphological and electron microscope observations showed the phenotype of increased lateral root under RNA methylation inhibitor (DZnepA) and DNA methylation inhibitor (5-azaC) treatment. RNA epitranscriptome based on Nanopore direct RNA sequencing revealed that DZnepA treatment exhibits significantly decreased m6A level in the 3'-untranslated region (3'-UTR), which was accompanied by increased gene expression, full-length ratio, higher proximal poly(A) site usage and shorter poly(A) tail length. DNA methylation levels of CG and CHG were reduced in both coding sequencing and transposable element upon 5-azaC treatment. Cell wall synthesis was impaired under methylation inhibition. In particular, differentially expressed genes showed a high percentage of overlap between DZnepA and 5-azaC treatment, which suggested a potential correlation between two methylations. This study provides preliminary information for a better understanding of the link between m6A and 5mC in root development of moso bamboo.