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Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. To produce a protein, a gene’s DNA is first copied to make molecules of messenger RNA (mRNA). The mRNAs pass through a molecular machine known as the ribosome, which translates the genetic code to make a protein. Not all of an mRNA is translated to make a protein; the “untranslated” regions play crucial roles in regulating how much of the protein is produced. In animals, plants and other eukaryotes, many mRNAs are made up of small pieces that are “spliced” together. During this process, proteins are deposited on the mRNA to mark the splice junctions, which are then cleared when the mRNA is translated. Many different mRNAs can be produced from the same gene by splicing different combinations of RNA pieces. Each of these mRNA “isoforms” can, in principle, contain a unique set of features that control its translation. Hence each mRNA isoform can be translated differently so that different amounts of the corresponding protein product are produced. However, the relationship between the variety of isoforms and the control of translation is complex and not well understood. To address these questions, Floor and Doudna measured the translation of over 60,000 mRNA isoforms made from almost 14,000 human genes. The experiments show that untranslated regions at the end of the mRNA (known as the 3′ end) strongly influence translation, even if the protein coding regions remain the same. Furthermore, the data showed that mRNAs with more splice junctions are translated better, implying an mRNA has some sort of memory of how many junctions it had even after the protein markers have been cleared. Next, Floor and Doudna inserted regulatory sequences from differently translated isoforms into an unrelated “reporter” gene. This dramatically changed the amount of protein produced from the reporter gene, in a manner predicted by the earlier experiments. Untranslated regions at the beginning of the mRNAs (known as the 5′ end) controlled the amount of protein produced from the reporter consistently across different types of cells from the body. On the other hand, the 3′ regions can tune the level of protein production in particular types of cells. Floor and Doudna’s findings demonstrate that differences between mRNA isoforms of a gene can have a big effect on the level of protein production. Changes in the types of mRNA made from a gene are often associated with human diseases, and these findings suggest one reason why. Additionally, the ability to engineer translation of an mRNA using the data is likely to aid the development of mRNA-based therapies.

Recent improvements in the accuracy of long-read sequencing (LRS) technologies have expanded the scope for novel transcriptional isoform discovery. Additionally, these advancements have improved the precision of transcript quantification, enabling a more accurate reconstruction of complex splicing patterns and transcriptomes. Thus, this project aims to take advantage of these analytical developments for the discovery and analysis of RNA isoforms in the human brain. A set of novel transcript isoforms was compiled using three bioinformatic tools, quantifying their expression across eight replicates of the cerebellar hemisphere, five replicates of the frontal cortex, and six replicates of the putamen. By taking a subset of the novel isoforms consistent across all discovery methods, a set of 170 highly confident novel RNA isoforms was curated for downstream analysis. This set consisted of 104 messenger RNAs (mRNAs) and 66 long non-coding RNAs (lncRNAs) isoforms. The detailed structure, expression, and potential encoded proteins of novel mRNA isoform BambuTx321 have been further described as an exemplary representative. Additionally, the tissue-specific expression [mean counts per million (CPM) of 5.979] of novel lncRNA, BambuTx1299, in the cerebellar hemisphere was observed. Overall, this project has identified and annotated several novel RNA isoforms across diverse tissues of the human brain, providing insights into their expression patterns and investigating their potential functional roles. Thus, this project has contributed to a more comprehensive understanding of the brain’s transcriptomic landscape for applications in basic research.

The western painted turtle, Chrysemys picta bellii , has the greatest tolerance to anoxia of any tetrapod studied to date. These turtles reside in the northern United States and southern Canada, and survive months of anoxia while submerged in ice-locked ponds and bogs. Reference genomes provide an important resource for elucidating the molecular bases for such unique physiological traits. The initial reference genome for this species, published in 2013, is highly fragmented, thereby limiting downstream analyses and biological interpretation. We created a new and improved assembly by combining PacBio HiFi, 10 × Genomics Chromium, Hi-C sequence data and Bionano optical mapping derived from a single individual to generate a new haplotype-resolved chromosome-level reference assembly for C. picta bellii : “SLU_Cpb5.0”. The genome size of the primary assembly is 2.372 Gb with a scaffold N50 of 133.6 Mb, a 6.5-fold improvement over the previous assembly. Annotation of SLU_Cpb5.0 revealed 12,242 novel genes compared to previous assemblies. PacBio Iso-Seq RNA sequencing using twelve tissues identified more than 100,000 novel transcript isoforms and 3,910 novel genes not previously annotated. This new genome assembly and annotation will support future comparative genomics studies, and the distinct patterns of tissue-specific isoform expression create a robust foundation for future characterization of the functions of these genes. Furthermore, to better understand the genetic basis of C. picta bellii ’s extreme physiological adaptations and other aspects of its biology, we utilize existing RNA-seq data to identify dozens of novel, differentially expressed genes in the heart and brain of anoxic painted turtles.

Background While numerous studies have described the transcriptomes of extracellular vesicles (EVs) in different cellular contexts, these efforts have typically relied on sequencing methods requiring RNA fragmentation, which limits interpretations on the integrity and isoform diversity of EV-targeted RNA populations. It has been assumed that mRNA signatures in EVs are likely to be fragmentation products of the cellular mRNA material, and the extent to which full-length mRNAs are present within EVs remains to be clarified. Results Using long-read nanopore RNA sequencing, we sought to characterize the full-length polyadenylated (poly-A) transcriptome of EVs released by human chronic myelogenous leukemia K562 cells. We detected 443 and 280 RNAs that were respectively enriched or depleted in EVs. EV-enriched poly-A transcripts consist of a variety of biotypes, including mRNAs, long non-coding RNAs, and pseudogenes. Our analysis revealed that 10.58% of all EV reads, and 18.67% of all cellular (WC) reads, corresponded to known full-length transcripts, with mRNAs representing the largest biotype for each group (EV = 58.13%, WC = 43.93%). We also observed that for many well-represented coding and non-coding genes, diverse full-length transcript isoforms were present in EV specimens, and these isoforms were reflective-of but often in different ratio compared to cellular samples. Conclusion This work provides novel insights into the compositional diversity of poly-A transcript isoforms enriched within EVs, while also underscoring the potential usefulness of nanopore sequencing to interrogate secreted RNA transcriptomes.

Human cone photoreceptors differ from rods and serve as the retinoblastoma cell-of-origin, yet the developmental basis for their distinct behaviors is poorly understood. Here, we used deep full-length single-cell RNA-sequencing (scRNA-seq) to distinguish post-mitotic cone and rod developmental states and identify cone-specific features related to retinoblastomagenesis. The analyses revealed nascent, immediately post-mitotic cone and rod precursors characterized by higher THRB or NRL regulon activities, immature and maturing cone and rod precursors with concurrent cone- and rod-related gene and regulon expression, and distinct early and late cone and rod maturation states distinguished by maturation-associated declines in RAX regulon activity. Cell-state-specific gene expression features inferred from full-length scRNA-seq were consistent with past 3' scRNA-seq analyses. Beyond the cell state characterizations, full-length scRNA-seq revealed that both L/M cone and rod precursors co-expressed NRL and THRB RNAs yet differentially expressed functionally antagonistic NRL isoforms and prematurely terminated THRB transcripts. Moreover, early L/M cone precursors sequentially expressed several lncRNAs along with MYCN , which composed the seventh most L/M-cone-specific regulon, and SYK , which was implicated in the cone precursors’ proliferative response to RB1 loss. These findings reveal previously unresolved photoreceptor precursor states and suggest a role for early cone-precursor-intrinsic SYK expression in retinoblastoma initiation.

We developed a single-cell approach to detect CRISPR-modified mRNA transcript structures. This method assesses how genetic variants at splicing sites and splicing factors contribute to alternative mRNA isoforms. We determine how alternative splicing is regulated by editing target exon-intron segments or splicing factors by CRISPR-Cas9 and their consequences on transcriptome profile. Our method combines long-read sequencing to characterize the transcript structure and short-read sequencing to match the single-cell gene expression profiles and gRNA sequence and therefore provides targeted genomic edits and transcript isoform structure detection at single-cell resolution.

Hox genes play a crucial role in morphogenesis, especially in anterior–posterior body axis patterning. The organization of Hox clusters in vertebrates is a result of several genome duplications: two rounds of duplication in the ancestors of all vertebrates and a third round that was specific for teleost fishes. Teleostei cluster structure has been significantly modified in the evolutionary processes by Hox gene losses and co-options, while mammals show no such tendency. In mammals, the Hox gene number in a single cluster is stable and generally large, and the numbers are similar to those in the Chondrichthyes. Hox gene alternative splicing activity slightly differs between fishes and mammals. Fishes and mammals have differences in their known alternative splicing activity for Hox gene distribution along the anterior–posterior body axis. The analyzed fish groups—the Coelacanthiformes, Chondrichthyes, and Teleostei—all have higher known alternative mRNA numbers from the anterior and posterior regions, whereas mammals have a more uniform Hox transcript distribution along this axis. In fishes, most Hox transcripts produce functioning proteins, whereas mammals have significantly more known transcripts that do not produce functioning proteins.

Background Cell type specialization is a hallmark of complex multicellular organisms and is usually established through implementation of cell-type-specific gene expression programs. The multicellular green alga Volvox carteri has just two cell types, germ and soma, that have previously been shown to have very different transcriptome compositions which match their specialized roles. Here we interrogated another potential mechanism for differentiation in V. carteri , cell type specific alternative transcript isoforms (CTSAI). Methods We used pre-existing predictions of alternative transcripts and de novo transcript assembly with HISAT2 and Ballgown software to compile a list of loci with two or more transcript isoforms, identified a small subset that were candidates for CTSAI, and manually curated this subset of genes to remove false positives. We experimentally verified three candidates using semi-quantitative RT-PCR to assess relative isoform abundance in each cell type. Results Of the 1978 loci with two or more predicted transcript isoforms 67 of these also showed cell type isoform expression biases. After curation 15 strong candidates for CTSAI were identified, three of which were experimentally verified, and their predicted gene product functions were evaluated in light of potential cell type specific roles. A comparison of genes with predicted alternative splicing from Chlamydomonas reinhardtii , a unicellular relative of V. carteri , identified little overlap between ortholog pairs with alternative splicing in both species. Finally, we interrogated cell type expression patterns of 126 V . carteri predicted RNA binding protein (RBP) encoding genes and found 40 that showed either somatic or germ cell expression bias. These RBPs are potential mediators of CTSAI in V. carteri and suggest possible pre-adaptation for cell type specific RNA processing and a potential path for generating CTSAI in the early ancestors of metazoans and plants. Conclusions We predicted numerous instances of alternative transcript isoforms in Volvox, only a small subset of which showed cell type specific isoform expression bias. However, the validated examples of CTSAI supported existing hypotheses about cell type specialization in V. carteri, and also suggested new hypotheses about mechanisms of functional specialization for their gene products. Our data imply that CTSAI operates as a minor but important component of V. carteri cellular differentiation and could be used as a model for how alternative isoforms emerge and co-evolve with cell type specialization.

IntroductionWhile tens of thousands of HLA alleles have been identified by DNA sequencing, the contribution of alternative splicing to HLA diversity is not well characterized. In this study, we sought to determine if long-read sequencing could be used to accurately quantify allele-specific HLA transcripts in primary human lymphocytes.MethodscDNA libraries were prepared from peripheral blood lymphocytes from 12 donors and sequenced by nanopore long-read sequencing. HLA reads were aligned to donor-specific reference sequences based on the known type of each donor. Allele-specific exon utilization was calculated as the proportion of reads aligning to each allele containing known exons, and transcript isoforms were quantified based on patterns of exon utilization within individual reads.ResultsSplice variants were rare among class I HLA genes (median exon retention rate 99%–100%), except for several HLA-C alleles with exon 5 spliced out of up to 15% of reads. Splice variants were also rare among class II HLA genes (median exon retention rate 98%–100%), except for HLA-DQB1 . Consistent with previous work, exon 5 of HLA-DQB1 was spliced out in alleles with a mutated splice acceptor site at rs28688207. Surprisingly, a 28% loss of exon 5 was also observed in HLA-DQB1 alleles with an intact splice acceptor site at rs28688207.DiscussionWe describe a simple bioinformatic workflow to quantify allele-specific expression of HLA transcript isoforms. Further studies are warranted to characterize the repertoire of HLA transcripts expressed in different cell types and tissues across diverse populations.

Alternative splicing (AS) is a crucial post-transcriptional regulatory mechanism that enhances transcriptomic and proteomic diversity by generating multiple mRNA isoforms from a single gene. In plants, AS plays a central role in modulating growth, development, and stress responses. We summarize the prevalence and functional roles of AS in plant development and stress adaptation, highlighting mechanisms that link AS to hormone signaling, RNA surveillance, and epigenetic regulation. Polyploid crops, with their duplicated genomes, exhibit expanded AS complexity, contributing to phenotypic plasticity, stress tolerance, and adaptive evolution. Thus, this review synthesizes current knowledge on AS in plants, with a focus on three economically important polyploid crops—Brassica napus, Gossypium hirsutum, and Triticum aestivum. We also discuss how subgenome interactions shape diversity in polyploids and influence trait variation. Despite significant advances enabled by high-throughput sequencing, mechanistic studies that directly link specific AS events to phenotypic outcomes remain limited. Understanding how polyploidy reprograms AS and how isoform variation contributes to stress adaptation will be critical for harnessing AS in crop improvement.