Regulation and function of alternative polyadenylation in development and differentiation.

Alternative processing of nascent mRNAs is widespread in eukaryotic organisms and greatly impacts the output of gene expression. Specifically, alternative cleavage and polyadenylation (APA) is a co-transcriptional molecular process that switches the polyadenylation site (PAS) at which a nascent mRNA is cleaved, resulting in mRNA isoforms with different 3'UTR length and content. APA can potentially affect mRNA translation efficiency, localization, stability, and mRNA seeded protein-protein interactions. APA naturally occurs during development and cellular differentiation, with around 70% of human genes displaying APA in particular tissues and cell types. For example, neurons tend to express mRNAs with long 3'UTRs due to preferential processing at PASs more distal than other PASs used in other cell types. In addition, changes in APA mark a variety of pathological states, including many types of cancer, in which mRNAs are preferentially cleaved at more proximal PASs, causing expression of mRNA isoforms with short 3'UTRs. Although APA has been widely reported, both the function of APA in development and the mechanisms that regulate the choice of 3'end cut sites in normal and pathogenic conditions are still poorly understood. In this review, we summarize current understanding of how APA is regulated during development and cellular differentiation and how the resulting change in 3'UTR content affects multiple aspects of gene expression. With APA being a widespread phenomenon, the advent of cutting-edge scientific techniques and the pressing need for in-vivo studies, there has never been a better time to delve into the intricate mechanisms of alternative cleavage and polyadenylation.

The emerging theme of 3'UTR mRNA isoform regulation in reprogramming of cell metabolism.

The 3' untranslated region (3'UTR) of mRNA plays a key role in the post-transcriptional regulation of gene expression. Most eukaryotic protein-coding genes express 3'UTR isoforms owing to alternative cleavage and polyadenylation (APA). The 3'UTR isoform expression profile of a cell changes in cell proliferation, differentiation, and stress conditions. Here, we review the emerging theme of regulation of 3'UTR isoforms in cell metabolic reprogramming, focusing on cell growth and autophagy responses through the mTOR pathway. We discuss regulatory events that converge on the Cleavage Factor I complex, a master regulator of APA in 3'UTRs, and recent understandings of isoform-specific m6A modification and endomembrane association in determining differential metabolic fates of 3'UTR isoforms.

Sites of transcription initiation drive mRNA isoform selection.

The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3' end site selection. Applying long-read sequencing to accurately represent even the longest transcripts from end to end, we quantify mRNA isoforms in Drosophila tissues, including the transcriptionally complex nervous system. We find that in Drosophila heads, as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription initiation (TSS). "Dominant promoters," characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as p300/CBP loss disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.

An ancient testis-specific IQ motif-containing H gene regulates specific transcript isoform expression during spermatogenesis.

Spermatogenic cells express more alternatively spliced RNAs than most whole tissues; however, the regulation of these events remains unclear. Here, we have characterized the function of a testis-specific IQ motif-containing H gene (Iqch) using a mutant mouse model. We found that Iqch is essential for the specific expression of RNA isoforms during spermatogenesis. Using immunohistochemistry of the testis, we noted that Iqch was expressed mainly in the nucleus of spermatocyte and spermatid, where IQCH appeared juxtaposed with SRRM2 and ERSP1 in the nuclear speckles, suggesting that interactions among these proteins regulate alternative splicing (AS). Using RNA-seq, we found that mutant Iqch produces alterations in gene expression, including the clear downregulation of testis-specific lncRNAs and protein-coding genes at the spermatid stage, and AS modifications - principally increased intron retention - resulting in complete male infertility. Interestingly, we identified previously unreported spliced transcripts in the wild-type testis, while mutant Iqch modified the expression and use of hundreds of RNA isoforms, favouring the expression of the canonical form. This suggests that Iqch is part of a splicing control mechanism, which is essential in germ cell biology.

Identification and characterization of the Doublesex gene and its mRNA isoforms in the brine shrimp Artemia franciscana.

Doublesex (DSX) proteins are members of the Doublesex/mab-3-related (DMRT) protein family and play crucial roles in sex determination and differentiation among the animal kingdom. In the present study, we identified two Doublesex (Dsx)-like mRNA isoforms in the brine shrimp Artemia franciscana (Kellogg 1906), which are generated by the combination of alternative promoters, alternative splicing and alternative polyadenylation. The two transcripts exhibited sex-biased enrichment, which we termed AfrDsxM and AfrDsxF. They share a common region which encodes an identical N-terminal DNA-binding (DM) domain. RT-qPCR analyses showed that AfrDsxM is dominantly expressed in male Artemia while AfrDsxF is specifically expressed in females. Expression levels of both isoforms increased along with the developmental stages of their respective sexes. RNA interference with dsRNA showed that the knockdown of AfrDsxM in male larvae led to the appearance of female traits including an ovary-like structure in the original male reproductive system and an elevated expression of vitellogenin. However, silencing of AfrDsxF induced no clear phenotypic change in female Artemia. These results indicated that the male AfrDSXM may act as inhibiting regulator upon the default female developmental mode in Artemia. Furthermore, electrophoretic mobility shift assay analyses revealed that the unique DM domain of AfrDSXs can specifically bind to promoter segments of potential downstream target genes like AfrVtg. These data show that AfrDSXs play crucial roles in regulating sexual development in Artemia, and further provide insight into the evolution of sex determination/differentiation in sexual organisms.

Bidirectional effects of voluntary exercise on the expression of Bdnf isoforms in the hippocampus of Hatano rat strains displaying different activity levels.

Brain-derived neurotrophic factor has functional mRNA isoforms, whose expression is assumed to mediate the beneficial effects of exercise in neuropsychiatric disorders. This study aims to reveal the mechanism of intensity-dependent effects of voluntary exercise, focusing on the expression of Bdnf mRNA isoforms in Hatano rats. Animals with different voluntary activity were housed in cages with a locked or unlocked wheel for 5 weeks. The expression levels of Bdnf isoforms and the corresponding coding sequences (CDS) were measured in the hippocampus using real-time polymerase chain reaction (PCR). We found that exercise increased the expression of Bdnf isoform containing exon 1 in the high-intensity-running strain and decreased the expressions of Bdnf exon 1, 3, 6, 7, 8, and 9a in mild-intensity-running animal. The expression of Bdnf CDS was increased by exercise in both strains. These results suggest that expressions of Bdnf isoforms depend on the intensities of voluntary exercise, but the involvement of subjects' genetic background could not be excluded. Our finding also implies that the bidirectional effects of exercise may not be mediated via the final product of Bdnf.

mRNA isoform balance in neuronal development and disease.

Balanced mRNA isoform diversity and abundance are spatially and temporally regulated throughout cellular differentiation. The proportion of expressed isoforms contributes to cell type specification and determines key properties of the differentiated cells. Neurons are unique cell types with intricate developmental programs, characteristic cellular morphologies, and electrophysiological potential. Neuron-specific gene expression programs establish these distinctive cellular characteristics and drive diversity among neuronal subtypes. Genes with neuron-specific alternative processing are enriched in key neuronal functions, including synaptic proteins, adhesion molecules, and scaffold proteins. Despite the similarity of neuronal gene expression programs, each neuronal subclass can be distinguished by unique alternative mRNA processing events. Alternative processing of developmentally important transcripts alters coding and regulatory information, including interaction domains, transcript stability, subcellular localization, and targeting by RNA binding proteins. Fine-tuning of mRNA processing is essential for neuronal activity and maintenance. Thus, the focus of neuronal RNA biology research is to dissect the transcriptomic mechanisms that underlie neuronal homeostasis, and consequently, predispose neuronal subtypes to disease. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.

A critical developmental window for ELAV/Hu-dependent mRNA signatures at the onset of neuronal differentiation.

Cell-type-specific gene regulatory programs are essential for cell differentiation and function. In animal neurons, the highly conserved ELAV/Hu family of proteins promotes alternative splicing and polyadenylation of mRNA precursors to create unique neuronal transcript isoforms. Here, we assess transcriptome profiles and neurogenesis success in Drosophila models engineered to express differing levels of ELAV activity in the course of development. We show that the ELAV-mediated establishment of a subset of neuronal mRNA isoforms at the onset of neuron differentiation constitutes a developmental bottleneck that cannot be overcome later by the nuclear activation of the paralog found in neurons (FNE). Loss of ELAV function outside of that critical time window results in neurological defects. We find that FNE, when activated early enough, can restore ELAV-dependent neuronal mRNA isoforms and fully rescue development. Our findings demonstrate the essential role of robust cellular strategies to maintain ELAV activity and intact neuronal signatures in neurogenesis and neuronal function.

Developmentally regulated alternate 3' end cleavage of nascent transcripts controls dynamic changes in protein expression in an adult stem cell lineage.

Alternative polyadenylation (APA) generates transcript isoforms that differ in the position of the 3' cleavage site, resulting in the production of mRNA isoforms with different length 3' UTRs. Although widespread, the role of APA in the biology of cells, tissues, and organisms has been controversial. We identified >500 Drosophila genes that express mRNA isoforms with a long 3' UTR in proliferating spermatogonia but a short 3' UTR in differentiating spermatocytes due to APA. We show that the stage-specific choice of the 3' end cleavage site can be regulated by the arrangement of a canonical polyadenylation signal (PAS) near the distal cleavage site but a variant or no recognizable PAS near the proximal cleavage site. The emergence of transcripts with shorter 3' UTRs in differentiating cells correlated with changes in expression of the encoded proteins, either from off in spermatogonia to on in spermatocytes or vice versa. Polysome gradient fractionation revealed >250 genes where the long 3' UTR versus short 3' UTR mRNA isoforms migrated differently, consistent with dramatic stage-specific changes in translation state. Thus, the developmentally regulated choice of an alternative site at which to make the 3' end cut that terminates nascent transcripts can profoundly affect the suite of proteins expressed as cells advance through sequential steps in a differentiation lineage.

ScisorWiz: visualizing differential isoform expression in single-cell long-read data.

SUMMARY: RNA isoforms contribute to the diverse functionality of the proteins they encode within the cell. Visualizing how isoform expression differs across cell types and brain regions can inform our understanding of disease and gain or loss of functionality caused by alternative splicing with potential negative impacts. However, the extent to which this occurs in specific cell types and brain regions is largely unknown. This is the kind of information that ScisorWiz plots can provide in an informative and easily communicable manner. ScisorWiz affords its user the opportunity to visualize specific genes across any number of cell types, and provides various sorting options for the user to gain different ways to understand their data. ScisorWiz provides a clear picture of differential isoform expression through various clustering methods and highlights features such as alternative exons and single-nucleotide variants. Tools like ScisorWiz are key for interpreting single-cell isoform sequencing data. This tool applies to any single-cell long-read RNA sequencing data in any cell type, tissue or species. AVAILABILITY AND IMPLEMENTATION: Source code is available at http://github.com/ans4013/ScisorWiz. No new data were generated for this publication. Data used to generate figures was sourced from GEO accession token GSE158450 and available on GitHub as example data.

Small genetic variation affecting mRNA isoforms associated with marbling and meat color in beef cattle.

The aim of this study was to identify mRNA isoforms and small genetic variants that may be affecting marbling and beef color in Nellore cattle. Longissimus thoracis muscle samples from 20 bulls with different phenotypes (out of 80 bulls set) for marbling (moderate (n = 10) and low (n = 10) groups) and beef color (desirable (n = 10) and undesirable (n = 9) group) traits were used to perform transcriptomic analysis using RNA sequencing. Fourteen and 15 mRNA isoforms were detected as differentially expressed (DE) (P-value ≤ 0.001) between divergent groups for marbling and meat color traits, respectively. Some of those DE mRNA isoforms have shown sites of splicing modified by small structural variants as single nucleotide variant (SNV), insertion, and/or deletion. Enrichment analysis identified metabolic pathways, such as O2/CO2 exchange in erythrocytes, tyrosine biosynthesis, and phenylalanine degradation. The results obtained suggest potential key regulatory genes associated with these economically important traits for the beef industry and for the consumer.

Flexible pri-miRNA structures enable tunable production of 5' isomiRs.

The Drosha cleavage of a pri-miRNA defines mature microRNA sequence. Drosha cleavage at alternative positions generates 5' isoforms (isomiRs) which have distinctive functions. To understand how pri-miRNA structures influence Drosha cleavage, we performed a systematic analysis of the maturation of endogenous pri-miRNAs and their variants both in vitro and in vivo. We show that in addition to previously known features, the overall structural flexibility of pri-miRNA impact Drosha cleavage fidelity. Internal loops and nearby G · U wobble pairs on the pri-miRNA stem induce the use of non-canonical cleavage sites by Drosha, resulting in 5' isomiR production. By analysing patient data deposited in the Cancer Genome Atlas, we provide evidence that alternative Drosha cleavage of pri-miRNAs is a tunable process that responds to the level of pri-miRNA-associated RNA-binding proteins. Together, our findings reveal that Drosha cleavage fidelity can be modulated by altering pri-miRNA structure, a potential mechanism underlying 5' isomiR biogenesis in tumours.[Figure: see text].

Suppression of premature transcription termination leads to reduced mRNA isoform diversity and neurodegeneration.

Tight regulation of mRNA isoform expression is essential for neuronal development, maintenance, and function; however, the repertoire of proteins that govern isoform composition and abundance remains incomplete. Here, we show that the RNA kinase CLP1 regulates mRNA isoform expression through suppression of proximal cleavage and polyadenylation. We found that human stem-cell-derived motor neurons without CLP1 or with the disease-associated CLP1 p.R140H variant had distinct patterns of RNA-polymerase-II-associated cleavage and polyadenylation complex proteins that correlated with polyadenylation site usage. These changes resulted in imbalanced mRNA isoform expression of long genes important for neuronal function that were recapitulated in vivo. Strikingly, we observed the same pattern of reduced mRNA isoform diversity in 3' end sequencing data from brain tissues of patients with neurodegenerative disease. Together, our results identify a previously uncharacterized role for CLP1 in mRNA 3' end formation and reveal an mRNA misprocessing signature in neurodegeneration that may suggest a common mechanism of disease.

Single-cell characterization of CRISPR-modified transcript isoforms with nanopore sequencing.

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.

Altered cell and RNA isoform diversity in aging Down syndrome brains.

Down syndrome (DS), trisomy of human chromosome 21 (HSA21), is characterized by lifelong cognitive impairments and the development of the neuropathological hallmarks of Alzheimer's disease (AD). The cellular and molecular modifications responsible for these effects are not understood. Here we performed single-nucleus RNA sequencing (snRNA-seq) employing both short- (Illumina) and long-read (Pacific Biosciences) sequencing technologies on a total of 29 DS and non-DS control prefrontal cortex samples. In DS, the ratio of inhibitory-to-excitatory neurons was significantly increased, which was not observed in previous reports examining sporadic AD. DS microglial transcriptomes displayed AD-related aging and activation signatures in advance of AD neuropathology, with increased microglial expression of C1q complement genes (associated with dendritic pruning) and the HSA21 transcription factor gene RUNX1 Long-read sequencing detected vast RNA isoform diversity within and among specific cell types, including numerous sequences that differed between DS and control brains. Notably, over 8,000 genes produced RNAs containing intra-exonic junctions, including amyloid precursor protein (APP) that had previously been associated with somatic gene recombination. These and related results illuminate large-scale cellular and transcriptomic alterations as features of the aging DS brain.

Mapping and modeling the genomic basis of differential RNA isoform expression at single-cell resolution with LR-Split-seq.

The rise in throughput and quality of long-read sequencing should allow unambiguous identification of full-length transcript isoforms. However, its application to single-cell RNA-seq has been limited by throughput and expense. Here we develop and characterize long-read Split-seq (LR-Split-seq), which uses combinatorial barcoding to sequence single cells with long reads. Applied to the C2C12 myogenic system, LR-split-seq associates isoforms to cell types with relative economy and design flexibility. We find widespread evidence of changing isoform expression during differentiation including alternative transcription start sites (TSS) and/or alternative internal exon usage. LR-Split-seq provides an affordable method for identifying cluster-specific isoforms in single cells.

Expression of myosin heavy chain isoform mRNA transcripts in the masseter and medial pterygoid muscles / Expresión de transcripciones de ARNm de isoforma de cadena pesada de miosina en los músculos masetero y pterigoideo medial

SUMMARY: Both the masseter and medial pterygoid muscles elevate the mandible, raising the lower jaw by acting simultaneously on the lateral and medial surfaces of the mandibular ramus. Nevertheless, electromyographic studies indicate that these muscles, as well as the superficial and deep heads of the masseter, act in a different way during mastication. We have analyzed by real time quantitative polymerase chain reaction (RT-qPCR) the expression of myosin heavy chain (MHC) isoforms in the masseter and medial pterygoid muscles in humans in order to identify possible differences in the expression patterns that may be related to functional differences identified with electromyography. Our findings indicate that the expression pattern of MHC isoforms in the two muscles is characteristic of fast and powerful phasic muscles. We have also observed a high percentage of expression of the MHC-IIx isoform and the expression of the MHC-M isoform at the mRNA level in both muscles, an isoform that does not translate into protein in the masticatory muscles of humans. The high percentage of expression of the MHC-IIx isoform in humans can be related to a high contractile speed of the masseter and medial pterygoid in humans. On the other hand, the low percentage of expression of the MHC-M isoform at the mRNA level in both muscles can be related to the complex evolutionary process that has reduced the size and force of the masticatory muscles in humans.

RESUMEN: Los músculos masetero y pterigoideo medial elevan la mandíbula actuando de forma simultánea sobre las caras lateral y medial de su rama. Sin embargo, los estudios electromiográficos indican que estos dos músculos actúan de forma diferente durante la masticación, de la misma forma que lo hacen las porciones superficial y profunda del músculo masetero. En el presente estudio hemos analizado mediante PCR en tiempo real la expresión de las isoformas de la cadena pesada de la miosina o myosin heavy chain (MHC) en los músculos masetero y pterigoideo medial en humanos, con la finalidad de identificar diferencias en los patrones de expresión que se puedan relacionar con las diferencias funcionales identificadas con la electromiografía. Nuestros resultados indican que el patrón de expresión de las isoformas de la MHC en los dos músculos es la característica de los músculos rápidos y potentes. También hemos observado un elevado porcentaje de expresión de la isoforma MHC-IIx y la expresión a nivel de ARNm de la isoforma MHC-M en los dos músculos, una isoforma que no se detecta a nivel de proteína en los músculos masticadores humanos. El elevado porcentaje de expresión de la isoforma MHC-IIx que hemos observado se puede relacionar con una elevada velocidad de contracción de los músculos masetero y pterigoideo medial en los humanos. Por otro lado, el bajo porcentaje de expresión de la isoforma MHC-M a nivel de ARNm en ambos músculos se puede relacionar con los procesos evolutivos complejos que han reducido el tamaño y la fuerza de los músculos masticadores en los humanos.

Multiple G-quadruplexes in the LMNA promoter regulate LMNA variant 6 transcription and promote colon cancer cell growth.

Lamin A/C proteins, major components of the nuclear lamina, are encoded by the LMNA gene. These proteins have multiple cellular functions, including DNA transcription and replication, chromatin organization, regulation of the cell cycle, and apoptosis. Mutations in LMNA are associated with a variety of diseases called laminopathies. LMNA has implications in cancer; however, its mechanisms of dysregulation in cancer cells are not yet fully understood. In this study, among the LMNA transcript variants, we focused on a transcriptional variant 6 (termed LMNA-V6), which contains unique 3 exons upstream of exon 1 of LMNA. The promoter region of LMNA-V6 formed multiple G-quadruplexes and increased its transcriptional activity. Moreover, LMNA-V6 negatively regulated other LMNA mRNA variants, lamin A and lamin C, via direct interaction with their promoter. Knockdown of LMNA-V6 decreased the proliferation of colon cancer cells, whereas overexpression of the unique 3 exons of LMNA-V6 increased cell growth. Furthermore, microarray gene expression profiling showed that alteration of LMNA-V6 levels influenced the expression of p53 in colon cancer cells. Taken together, the results suggest that LMNA-V6 may be a novel functional RNA whose expression is regulated through multiple G-quadruplexes in colon cancer cells.

A ligand-insensitive UNC5B splicing isoform regulates angiogenesis by promoting apoptosis.

The Netrin-1 receptor UNC5B is an axon guidance regulator that is also expressed in endothelial cells (ECs), where it finely controls developmental and tumor angiogenesis. In the absence of Netrin-1, UNC5B induces apoptosis that is blocked upon Netrin-1 binding. Here, we identify an UNC5B splicing isoform (called UNC5B-Δ8) expressed exclusively by ECs and generated through exon skipping by NOVA2, an alternative splicing factor regulating vascular development. We show that UNC5B-Δ8 is a constitutively pro-apoptotic splicing isoform insensitive to Netrin-1 and required for specific blood vessel development in an apoptosis-dependent manner. Like NOVA2, UNC5B-Δ8 is aberrantly expressed in colon cancer vasculature where its expression correlates with tumor angiogenesis and poor patient outcome. Collectively, our data identify a mechanism controlling UNC5B's necessary apoptotic function in ECs and suggest that the NOVA2/UNC5B circuit represents a post-transcriptional pathway regulating angiogenesis.

Differential expression of Dp71 and Dp40 isoforms in proliferating and differentiated neural stem cells: Identification of Dp40 splicing variants.

Dp71 and Dp40 are the main products of the DMD gene in the central nervous system, and they are developmentally regulated from the early stages of embryonic development to adulthood. To further study the roles of Dp71 and Dp40 during cell proliferation and neural differentiation, we analyzed Dp71/Dp40 isoform expression at the mRNA level by RT-PCR assays to identify alternative splicing (AS) in the isoforms expressed in rat neural stem/progenitor cells (NSPCs) and in differentiated cells (neurons and glia). We found that proliferating NSPCs expressed Dp71d, Dp71dΔ71, Dp71f, Dp71fΔ71, Dp71dΔ74 and Dp40, as well as two Dp40 isoforms: Dp40Δ63,64 and Dp40Δ64-67. In differentiated cells we also found the expression of Dp71d, Dp71dΔ71, Dp71f, Dp71fΔ71 and Dp40. However, the expression frequencies were different in both stages. In addition, in differentiated cells, we found Dp71fΔ71-74, and interestingly, we did not find the expression of Dp71dΔ74 or the newly identified Dp40 isoforms. In this work we show that NSPC differentiation is accompanied by changes in Dp71/Dp40 isoform expression, suggesting different roles for these isoforms in NSPCs proliferation and neuronal differentiation, and we describe, for the first time, alternative splicing of Dp40.