mRNA Isoforms Research Articles

Identification of pennaceous barbule cell factor (PBCF), a novel gene with spatiotemporal expression in barbule cells during feather development.

Bird contour feathers exhibit a complex hierarchical structure composed of a rachis, barbs, and barbules, with barbules playing a crucial role in maintaining feather structure and function. Understanding the molecular mechanisms underlying barbule formation is essential for advancing our knowledge of avian biology and evolution. In this study, we identified a novel gene, pennaceous barbule cell factor (PBCF), using microarray analysis, RT-PCR, and in situ hybridization. PBCF is expressed in barbule cells adjacent to the ramus during pennaceous barbule formation, where these cells fuse with the ramus to establish the feather's branching structure. PBCF expression occurs transiently after melanin pigmentation of the barbule plates but before the expression of barbule-specific keratin 1 (BlSK1). Orthologues of PBCF, predicted to be secreted proteins, are conserved across avian species, with potential homologues detected in reptiles, suggesting an evolutionary lineage-specific adaptation. Additionally, PBCF is expressed in non-vacuolated notochord cells and the extra-embryonic ectoderm of the yolk sac, hinting at its broader developmental significance. The PBCF gene produces two mRNA isoforms via alternative splicing, encoding a secreted protein and a glycophosphatidylinositol (GPI)-anchored membrane-bound protein, indicating functional versatility. These findings suggest that PBCF may be involved as an avian-specific extracellular matrix component in cell adhesion and/or communication, potentially contributing to both feather development and embryogenesis. Further investigation of PBCF's role in feather evolution and its potential functions in other vertebrates could provide new insights into the interplay between development and evolution.

Differentially expressed messenger RNA isoforms in beef cattle skeletal muscle with different fatty acid profiles.

This study aimed to identify mRNA isoforms that were expressed differently in the muscle tissue of Nellore cattle based on their intramuscular fatty acid profile. Forty-eight young bulls were used to quantify beef fatty acids (FA) and perform RNA sequencing analysis. The young bulls were divided into three different groups based on quantifying FA using k-means analysis. The Grp1 clustered animals with significantly elevated levels of PUFA, ω6, ω3, linoleic acid, α-linolenic acid, and PUFA/SFA ratios, indicating a more favorable fatty acid profile. Animals in Group 3 demonstrated significantly higher levels of palmitic acid, stearic acid, myristic acid, and SFA, which are less favorable fatty acid profiles. Grp2 included bulls with intermediate levels of fatty acids, positioned between the profiles of Grp1 and Grp3. Differential expression (DE) analyses were performed to compare these three distinct groups through the contrasts: Grp1 vs. Grp2, Grp1 vs. Grp3, and Grp2 vs. Grp3. The DE analyses identified a total of 62, 26, and 30 transcripts for the contrasts Grp1 vs. Grp2, Grp1 vs. Grp3, and Grp2 vs. Grp3, respectively. In the comparison between the Grp1 and Grp2 groups, we identified three mRNA isoforms, C7-203, ADD1-204, and OXT-201, which are involved in glycogen and lipid metabolism. These mRNA isoforms regulate the key genes responsible for fatty acid synthesis, leading to a higher PUFA content profile. On the other hand, in the comparison between the Grp1 and Grp3 groups, the mRNA isoforms RBM3-202 and TRAG1-202 were identified and play a crucial role in muscle development, adipogenesis, and concentration of PUFA and MUFA, respectively. The downregulation of THRSP-201 and FABP4-201, isoforms identified in both, Grp1 vs. Grp2 and Grp2 vs. Grp3, contrasts may contribute to lower levels of MUFA and SFA and higher levels of PUFA. In addition, these mRNA isoforms were associated with lipogenesis, fatty acid transport, and inhibition of lipolysis. Our findings suggest that the identified mRNA isoforms could serve as promising candidates to help develop strategies to select animals of higher nutritional meat quality.

RNA editing produces mRNA isoforms that differ in stability and may broaden thermal tolerance range of mRNA and proteins of mussels <i>Mytilus</i>

RNA editing produces mRNA isoforms that differ in stability and may broaden thermal tolerance range of mRNA and proteins of mussels <i>Mytilus</i>

Direct and indirect effects of spliceosome disruption compromise gene regulation by Nonsense-Mediated mRNA Decay.

Pre-mRNA splicing, carried out in the nucleus by a large ribonucleoprotein machine known as the spliceosome, is functionally and physically coupled to the mRNA surveillance pathway in the cytoplasm called nonsense mediated mRNA decay (NMD). The NMD pathway monitors for premature translation termination signals, which can result from alternative splicing, by relying on the exon junction complex (EJC) deposited on exon-exon junctions by the spliceosome. Recently, multiple genetic screens in human cell lines have identified numerous spliceosome components as putative NMD factors. Using publicly available RNA-seq datasets from K562 and HepG2 cells depleted of 18 different spliceosome components, we find that natural NMD targeted mRNA isoforms are upregulated when members of the catalytic spliceosome are reduced. While some of this increase could be due to widespread pleiotropic effects of spliceosome dysfunction (e.g., reduced expression of NMD factors due to mis-splicing of their mRNAs), we identify that AQR, SF3B1, SF3B4 and CDC40 may have a more direct role in NMD. We also test the hypothesis that increased production of novel NMD substrates may overwhelm the pathway to find a direct correlation between the amount of novel NMD substrates detected and the degree of NMD inhibition observed. Finally, similar transcriptome alterations and NMD substrate upregulation are also observed in cells treated with spliceosome inhibitors and in cells derived from retinitis pigmentosa patients with mutations in PRPF8 and PRPF31. Overall, our results show that regardless of the cause, spliceosome disruption upregulates a broad set of NMD targets, which could contribute to cellular dysfunction in spliceosomopathies.

Two Novel Mouse Models of Duchenne Muscular Dystrophy with Similar Dmd Exon 51 Frameshift Mutations and Varied Phenotype Severity

Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disorder caused by an array of mutations in the dystrophin gene, with the most commonly mutated regions being exons 48–55. One of the several existing approaches to treat DMD is gene therapy, based on alternative splicing and mutant exon skipping. Testing of such therapy requires animal models that carry mutations homologous to those found in human patients. Here, we report the generation of two genetically modified mouse lines, named “insT” and “insG”, with distinct mutations at the same position in exon 51 that lead to a frameshift, presumably causing protein truncation. Hemizygous males of both lines exhibit classical signs of muscular dystrophy in all muscle tissues except for the cardiac tissue. However, pathological changes are more pronounced in one of the lines. Membrane localization of the protein is reduced to the point of absence in one of the lines. Moreover, an increase in full-length isoform mRNA was detected in diaphragms of insG line mice. Although further work is needed to qualify these mutations as sole origins of dissimilarity, both genetically modified mouse lines are suitable models of DMD and can be used to test gene therapy based on alternative splicing.

MRNA m5C Alteration in Azacitidine Demethylation Treatment of Acute Myeloid Leukemia.

The DNA demethylating therapy with azacitidine (AZA) is a promising therapeutic strategy for elderly patients with acute myeloid leukemia (AML). AZA primarily inhibits DNA methylation, promotes cell differentiation and apoptosis in AML. However, as a cytosine nucleoside analog, AZA also has the potential to be incorporated into RNA molecules. To assess the impact of AZA on RNA m5C methylation during demethylating therapy, we conducted Nanopore direct-RNA sequencing on samples from three AML patients pre and after demethylating therapy, as well as on HL-60 cells pretreated with AZA. We performed an integrated analysis of the transcriptome and the m5C methylome, contrasting the states of complete remission with those of active disease (AML). Our results revealed an extensive demethylation effect at the RNA level attributable to AZA and found that mRNA m5C modification may play a pivotal role in the progression of AML. Additionally, S100P was identified as a biomarker with significant prognostic implications. We also conducted a conjoint analysis of the transcriptome and the m5C methylome of the full-length transcripts, uncovering several dysregulated mRNA isoforms. Collectively, our findings indicate that mRNA m5C methylation is implicated during AML progression, and AZA exhibits an overall suppressive effect on this process.

Alternative splicing induces sample-level variation in gene–gene correlations

BackgroundThe vast majority of genes in the genome are multi-exonic, and are alternatively spliced during transcription, resulting in multiple isoforms for each gene. For some genes, different mRNA isoforms may have differential expression levels or be involved in different pathways. Bulk tissue RNA-seq, as a widely used technology for transcriptome quantification, measures the total expression (TE) levels of each gene across multiple isoforms in multiple cell types for each tissue sample. With recent developments in precise quantification of alternative splicing events for each gene, we propose to study the effects of alternative splicing variation on gene–gene correlation effects. We adopted a variance-component model for testing the TE–TE correlations of one gene with a co-expressed gene, accounting for the effects of splicing variation and splicing-by-TE interaction of one gene on the other.ResultsWe analyzed data from the Genotype-Tissue Expression (GTEx) project (V8). At the 5% FDR level, 38,146 pairs of genes out of ∼10 M examined pairs from GTEx lung tissue showed significant TE-splicing interaction effects, implying isoform-specific and/or sample-specific TE–TE correlations. Additional analysis across 13 GTEx brain tissues revealed strong tissue-specificity of TE-splicing interaction effects. Moreover, we showed that accounting for splicing variation across samples could improve the reproducibility of results and could reduce potential confounding effects in studying co-expressed gene pairs with bulk tissue data. Many of those gene pairs had correlation effects specific to only certain isoforms and would otherwise be undetected. By analyzing gene–gene co-expression variation within functional pathways accounting for splicing, we characterized the patterns of the “hub” genes with isoform-specific regulatory effects on multiple other genes.ConclusionsWe showed that splicing variation of a gene may interact with TE of the gene and affect the TE of co-expressed genes, resulting in substantial tissue-specific inter-sample variability in gene–gene correlation effects. Accounting for TE-splicing interaction effects could reduce potential confounding effects and improve the robustness of estimation when estimating gene–gene correlations from bulk tissue expression data.

THUMPD3 regulates alternative splicing of ECM transcripts in human lung cancer cells and promotes proliferation and migration.

RNA-modifying enzymes have recently garnered considerable attention due to their relevance in cancer biology, identifying them as potential targets for novel therapeutic intervention. THUMPD3 was recently identified as an RNA methyltransferase catalysing N2-methylguanosine (m2G) within certain tRNAs. In this study, we unveil a novel role for THUMPD3 in lung cancer cells. Depletion of the enzyme from lung cancer cells significantly impairs their fitness, negatively impacting key cellular processes such as proliferation and migration. Notably, exogenous expression of THUMPD3 in normal lung fibroblasts stimulates their proliferation rate. Additionally, transcriptome-wide analyses reveal that depletion of THUMPD3 from lung cancer cells induces substantial changes in the expression of cell surface proteins, including those comprising the extracellular matrix (ECM). We further demonstrate that THUMPD3 maintains expression of an extra-domain B (EDB) containing pro-tumour isoform of Fibronectin-1 mRNA, encoding FN1, an important ECM protein. Crucially, depletion of THUMPD3 promotes an alternative splicing event that removes the EDB-encoding exon from Fibronectin-1. This is consistent with THUMPD3 depletion reducing cellular proliferation and migration. Moreover, depletion of THUMPD3 selectively and preferentially affects the alternative splicing of ECM and cell adhesion molecule encoding transcripts, as well as those encoding neurodevelopmental proteins. Overall, these findings highlight THUMPD3 as an important player in regulating cancer-relevant alternative splicing and they provide a rationale for further investigations into THUMPD3 as a candidate target in anti-cancer therapy.

High-resolution profiling reveals coupled transcriptional and translational regulation of transgenes.

Concentrations of RNAs and proteins provide important determinants of cell fate. Robust gene circuit design requires an understanding of how the combined actions of individual genetic components influence both mRNA and protein levels. Here, we simultaneously measure mRNA and protein levels in single cells using HCR Flow-FISH for a set of commonly used synthetic promoters. We find that promoters generate differences in both the mRNA abundance and the effective translation rate of these transcripts. Stronger promoters not only transcribe more RNA but also show higher effective translation rates. While the strength of the promoter is largely preserved upon genome integration with identical elements, the choice of polyadenylation signal and coding sequence can generate large differences in the profiles of the mRNAs and proteins. We used long-read direct RNA sequencing to characterize full-length mRNA isoforms and observe remarkable uniformity of mRNA isoforms from the transgenic units. Together, our high-resolution profiling of transgenic mRNAs and proteins offers insight into the impact of common synthetic genetic components on transcriptional and translational mechanisms. By developing a novel framework for quantifying expression profiles of transgenes, we have established a system for comparing native and synthetic gene regulation and for building more robust transgenic systems.

Identification and characterization of human KALRN mRNA and Kalirin protein isoforms.

Kalirin is a multidomain protein with important roles in neurite outgrowth, and synaptic spine formation and remodeling. Genetic and pathophysiological links with various neuropsychiatric disorders associated with synaptic dysfunction and cognitive impairment have sparked interest in its potential as a pharmacological target. Multiple Kalirin proteoforms are detected in the adult human brain, yet we know little about the diversity of the transcripts that encode them or their tissue profiles. Here, we characterized full-length KALRN transcripts expressed in the adult human frontal lobe and hippocampus using rapid amplification of complementary DNA (cDNA) ends and nanopore long-read sequencing. For comparison with non-neural tissue, we also analyzed KALRN transcripts in the aorta. Multiple novel isoforms were identified and were largely similar between the two brain regions analyzed. Alternative splicing in the brain results in preferential inclusion of exon 37, which encodes 32 amino acids upstream of the second guanine nucleotide exchange factor (GEF) domain. Structural modeling predicts that a subset of these amino acids forms a conserved alpha helix. Although deletion of these amino acids had little effect on GEF activity, it did alter Kalirin-induced neurite outgrowth suggesting that this brain-enriched splicing event may be important for neural function. These data indicate that alternative splicing is potentially important for regulating Kalirin actions in the human brain.

RNA Isoform Diversity in Human Neurodegenerative Diseases.

Single-nucleus RNA-sequencing (snRNA-seq) has revealed new levels of cellular organization and diversity within the human brain. However, full-length mRNA isoforms are not resolved in typical snRNA-seq analyses using short-read sequencing that cannot capture full-length transcripts. Here we combine standard 10x Genomics short-read snRNA-seq with targeted PacBio long-read snRNA-seq to examine isoforms of genes associated with neurological diseases at the single-cell level from prefrontal cortex samples of diseased and nondiseased human brain, assessing over 165,000 cells. Samples from 25 postmortem donors with Alzheimer's disease (AD), dementia with Lewy bodies (DLB), or Parkinson's disease (PD), along with age-matched controls, were compared. Analysis of the short-read libraries identified shared and distinct gene expression changes across the diseases. The same libraries were then assayed using enrichment probes to target 50 disease-related genes followed by long-read PacBio sequencing, enabling linkage between cell type and isoform expression. Vast mRNA isoform diversity was observed in all 50 targeted genes, even those that were not differentially expressed in the short-read data. We also developed an informatics method for detection of isoform structural differences in novel isoforms versus the reference annotation. These data expand available single-cell datasets of the human prefrontal cortical transcriptome with combined short- and long-read sequencing across AD, DLB, and PD, revealing increased mRNA isoform diversity that may contribute to disease features and could potentially represent therapeutic targets for neurodegenerative diseases.

Alternative polyadenylation in cancer: Molecular mechanisms and clinical application.

Alternative polyadenylation (APA) serves as a crucial mechanism for the posttranscriptional regulation of gene expression and influences gene expression by generating diverse mRNA isoforms. This process is regulated by a diverse array of RNA-binding proteins (RBPs), which selectively bind to specific sequences or structures within the pre-mRNA molecule. Dysregulation of APA and its associated RBPs has been implicated in numerous diseases, including cardiovascular diseases, nervous system disease, and cancer. For instance, aberrant APA events have been observed in several types of tumors, contributing to tumor heterogeneity and affecting key cellular pathways involved in cell proliferation, invasion, metastasis, and response to therapy. This review critically evaluates the current understanding of APA mechanisms and the multifaceted roles of RBPs in orchestrating this intricate process. We highlight recent advancements in high-throughput sequencing and bioinformatics tools that have enhanced our ability to study APA on a genome-wide scale. Moreover, we explored the pathological consequences of APA dysregulation, emphasizing its role in oncogenesis. By elucidating the intricate relationships between APA and RBPs, this review aims to underscore the potential of targeting the APA machinery and RBPs for therapeutic intervention. Understanding these molecular processes holds promise for developing novel diagnostic markers and treatment strategies for a range of human cancers.

Generation of Transcript Length Variants and Reprogramming of mRNA Splicing During Atherosclerosis Progression in ApoE-Deficient Mice.

Background. Variant 3'UTRs provide mRNAs with different binding sites for miRNAs or RNA-binding proteins (RBPs) allowing the establishment of new regulatory environments. Regulation of 3'UTR length impacts on the control of gene expression by regulating accessibility of miRNAs or RBPs to homologous sequences in mRNAs. Objective. Studying the dynamics of mRNA length variations in atherosclerosis (ATS) progression and reversion in ApoE-deficient mice exposed to a high-fat diet and treated with an αCD40-specific siRNA or with a sequence-scrambled siRNA as control. Methods. We gathered microarray mRNA expression data from the aortas of mice after 2 or 16 weeks of treatments, and used these data in a Bioinformatics analysis. Results. Here, we report the lengthening of the 5'UTR/3'UTRs and the shortening of the CDS in downregulated mRNAs during ATS progression. Furthermore, treatment with the αCD40-specific siRNA resulted in the partial reversion of the 3'UTR lengthening. Exon analysis showed that these length variations were actually due to changes in the number of exons embedded in mRNAs, and the further examination of transcripts co-expressed at weeks 2 and 16 in mice treated with the control siRNA revealed a process of mRNA isoform switching in which transcript variants differed in the patterns of alternative splicing or activated latent/cryptic splice sites. Conclusion. We document length variations in the 5'UTR/3'UTR and CDS of mRNAs downregulated during atherosclerosis progression and suggest a role for mRNA splicing reprogramming and transcript isoform switching in the generation of disease-related mRNA sequence diversity and variability.

EIF3 engages with 3'-UTR termini of highly translated mRNAs.

Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3' untranslated region (3'-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3'-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3'-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling, but not with translational efficiency. The results presented here show that eIF3 engages with 3'-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.

Nutrient control of growth and metabolism through mTORC1 regulation of mRNA splicing

Cellular growth and organismal development are remarkably complex processes that require the nutrient-responsive kinase mechanistic target of rapamycin complex 1 (mTORC1). Anticipating that important mTORC1 functions remained to be identified, we employed genetic and bioinformatic screening in C.elegans to uncover mechanisms of mTORC1 action. Here, we show that during larval growth, nutrients induce an extensive reprogramming of gene expression and alternative mRNA splicing by acting through mTORC1. mTORC1 regulates mRNA splicing and the production of protein-coding mRNA isoforms largely independently of its target p70 S6 kinase (S6K) by increasing the activity of the serine/arginine-rich (SR) protein RSP-6 (SRSF3/7) and other splicing factors. mTORC1-mediated mRNA splicing regulation is critical for growth; mediates nutrient control of mechanisms that include energy, nucleotide, amino acid, and other metabolic pathways; and may be conserved in humans. Although mTORC1 inhibition delays aging, mTORC1-induced mRNA splicing promotes longevity, suggesting that when mTORC1 is inhibited, enhancement of this splicing might provide additional anti-aging benefits.

Assessment of the Concentration of Transforming Growth Factor Beta 1-3 in Degenerated Intervertebral Discs of the Lumbosacral Region of the Spine.

The purpose of this study was to evaluate the feasibility of using the expression profile of transforming growth factor beta (TGF-β-1-3) to assess the progression of L/S spine degenerative disease. The study group consisted of 113 lumbosacral (L/S) intervertebral disc (IVD) degenerative disease patients from whom IVDs were collected during a microdiscectomy, whereas the control group consisted of 81 participants from whom IVDs were collected during a forensic autopsy or organ harvesting. Hematoxylin and eosin staining was performed to exclude degenerative changes in the IVDs collected from the control group. The molecular analysis consisted of reverse-transcription real-time quantitative polymerase chain reaction (RT-qPCR), an enzyme-linked immunosorbent assay (ELISA), Western blotting, and an immunohistochemical analysis (IHC). In degenerated IVDs, we noted an overexpression of all TGF-β-1-3 mRNA isoforms with the largest changes observed for TGF-β3 isoforms (fold change (FC) = 19.52 ± 2.87) and the smallest for TGF-β2 (FC = 2.26 ± 0.16). Changes in the transcriptional activity of TGF-β-1-3 were statistically significant (p < 0.05). Significantly higher concentrations of TGF-β1 (2797 ± 132 pg/mL vs. 276 ± 19 pg/mL; p < 0.05), TGF-β2 (1918 ± 176 pg/mL vs. 159 ± 17 pg/mL; p < 0.05), and TGF-β3 (2573 ± 102 pg/mL vs. 152 ± 11 pg/mL) were observed in degenerative IVDs compared with the control samples. Determining the concentration profiles of TGF-β1-3 appears to be a promising monitoring tool for the progression of degenerative disease as well as for evaluating its treatment or developing new treatment strategies with molecular targets.

Post-transcriptional control drives Aurora kinase A expression in human cancers.

Aurora kinase A (AURKA) is a major regulator of the cell cycle. A prominent association exists between high expression of AURKA and cancer, and impairment of AURKA levels can trigger its oncogenic activity. In order to explore the contribution of post-transcriptional regulation to AURKA expression in different cancers, we carried out a meta-analysis of -omics data of 18 cancer types from The Cancer Genome Atlas (TCGA). Our study confirmed a general trend for increased AURKA mRNA in cancer compared to normal tissues and revealed that AURKA expression is highly dependent on post-transcriptional control in several cancers. Correlation and clustering analyses of AURKA mRNA and protein expression, and expression of AURKA-targeting hsa-let-7a miRNA, unveiled that hsa-let-7a is likely involved to varying extents in controlling AURKA expression in cancers. We then measured differences in the short/long ratio (SLR) of the two alternative cleavage and polyadenylation (APA) isoforms of AURKA mRNA across cancers compared to the respective healthy counterparts. We suggest that the interplay between APA and hsa-let-7a targeting of AURKA mRNA may influence AURKA expression in some cancers. hsa-let-7a and APA may also independently contribute to altered AURKA levels. Therefore, we argue that AURKA mRNA and protein expression are often discordant in cancer as a result of dynamic post-transcriptional regulation.

BIOM-35. COMPREHENSIVE IDENTIFICATION OF MRNA ISOFORMS AND THEIR DIAGNOSTIC POTENTIAL IN GLIOMAS

Abstract Most genes generate multiple mRNA isoforms which may differ in their protein-coding capacity. Also, cancer and its prognosis are associated with the altered expression of specific isoforms of a variety of genes as cancer cells can manipulate regulatory mechanisms to express isoforms that provide a survival advantage and/or resistance to therapy. However, the underlying mechanisms behind their pathology are poorly understood. Fifty seven glioma samples that could be categorized unambiguously into Astrocytoma (n=17), Oligodendroglioma (n=16) and Glioblastoma (n=24) based on pathology reports and according to WHO standards were selected for this analysis. Total RNA-Sequencing (RNA-Seq) was performed on RNA isolated from these 57 samples. RNA-Seq data analysis was performed using the DRAGEN RNA application in Illumina BaseSpace and transcript counts were generated after alignment. From these counts, isoforms that were significantly differentially expressed in Astrocytoma (Astro) vs Oligodendroglioma (Oligo), and Astrocytoma vs Glioblastoma (GBM) were identified using the R package, IsoformSwitchAnalyzeR. Approximately 93,000 transcripts were screened in both comparisons of which 4388 and 44 transcript switches were identified as significant at FDR&amp;lt;0.05 in the Astro vs GBM and Astro vs Oligo comparisons, respectively. Applying an additional cut-off based on the difference in isoform fraction or dIF of a transcript between the two groups being compared (dIF&amp;gt;1.0 and dIF&amp;lt;-0.1), the most significant top switches were identified.. Similarly, in the Astro vs GBM comparison, the isoform switch in genes such as MDF1 and RER1 whose overexpression is known to be involved in tumorigenesis and metastasis were among the 155 top switches. Among the six top switches in the Astro vs. Oligo comparison were transcripts of the RENBP and GCNT2 genes which play an important role in the progression of melanoma, gastric and lung cancers. These results indicate that there may be isoform-specific dysregulation within gliomas, and further suggest that their biological significance and diagnostic potential can be explored.

Predicting synthetic mRNA stability using massively parallel kinetic measurements, biophysical modeling, and machine learning.

mRNA degradation is a central process that affects all gene expression levels, though it remains challenging to predict the stability of a mRNA from its sequence, due to the many coupled interactions that control degradation rate. Here, we carried out massively parallel kinetic decay measurements on over 50,000 bacterial mRNAs, using a learn-by-design approach to develop and validate a predictive sequence-to-function model of mRNA stability. mRNAs were designed to systematically vary translation rates, secondary structures, sequence compositions, G-quadruplexes, i-motifs, and RppH activity, resulting in mRNA half-lives from about 20 seconds to 20 minutes. We combined biophysical models and machine learning to develop steady-state and kinetic decay models of mRNA stability with high accuracy and generalizability, utilizing transcription rate models to identify mRNA isoforms and translation rate models to calculate ribosome protection. Overall, the developed model quantifies the key interactions that collectively control mRNA stability in bacterial operons and predicts how changing mRNA sequence alters mRNA stability, which is important when studying and engineering bacterial genetic systems.

Challenges in identifying mRNA transcript starts and ends from long-read sequencing data.

Long-read sequencing (LRS) technologies have the potential to revolutionize scientific discoveries in RNA biology through the comprehensive identification and quantification of full-length mRNA isoforms. Despite great promise, challenges remain in the widespread implementation of LRS technologies for RNA-based applications, including concerns about low coverage, high sequencing error, and robust computational pipelines. Although much focus has been placed on defining mRNA exon composition and structure with LRS data, less careful characterization has been done of the ability to assess the terminal ends of isoforms, specifically, transcription start and end sites. Such characterization is crucial for completely delineating full mRNA molecules and regulatory consequences. However, there are substantial inconsistencies in both start and end coordinates of LRS reads spanning a gene, such that LRS reads often fail to accurately recapitulate annotated or empirically derived terminal ends of mRNA molecules. Here, we describe the specific challenges of identifying and quantifying mRNA terminal ends with LRS technologies and how these issues influence biological interpretations of LRS data. We then review recent experimental and computational advances designed to alleviate these problems, with ideal use cases for each approach. Finally, we outline anticipated developments and necessary improvements for the characterization of terminal ends from LRS data.