(Alternative) transcription start sites as regulators of RNA processing.

Alternative transcription start site usage (ATSS) is a widespread regulatory strategy that enables genes to choose between multiple genomic loci for initiating transcription. This mechanism is tightly controlled during development and is often altered in disease states. In this review, we examine the growing evidence highlighting a role for transcription start sites (TSSs) in the regulation of mRNA isoform selection during and after transcription. We discuss how the choice of transcription initiation sites influences RNA processing and the importance of this crosstalk for cell identity and organism function. We also speculate on possible mechanisms underlying the integration of transcriptional and post-transcriptional processes.

Identification of regulatory links between transcription and RNA processing with long-read sequencing.

We present a detailed protocol for sequencing full-length mRNA isoforms using the Oxford nanopore long-read sequencing technology. We describe steps for poly(A) RNA isolation, library preparation, and cDNA size selection. We then detail procedures for sequencing and processing and a computational framework to identify exon couplings and assign mRNA 5' ends and 3' ends to each other. Our approach enables the identification of links between transcription initiation and co-transcriptional RNA processing events. For complete details on the use and execution of this protocol, please refer to Alfonso-Gonzalez et al.1.

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.

Regulation of neuronal RNA signatures by ELAV/Hu proteins.

The RNA-binding proteins encoded by the highly conserved elav/Hu gene family, found in all metazoans, regulate the expression of a wide range of genes, at both the co-transcriptional and posttranscriptional level. Nervous-system-specific ELAV/Hu proteins are prominent for their essential role in neuron differentiation, and mutations have been associated with human neurodevelopmental and neurodegenerative diseases. Drosophila ELAV, the founding member of the protein family, mediates the synthesis of neuronal RNA signatures by promoting alternative splicing and alternative polyadenylation of hundreds of genes. The recent identification of ELAV's direct RNA targets revealed the protein's central role in shaping the neuronal transcriptome, and highlighted the importance of neuronal transcript signatures for neuron maintenance and organism survival. Animals have evolved multiple cellular mechanisms to ensure robustness of ELAV/Hu function. In Drosophila, elav autoregulates in a 3'UTR-dependent manner to maintain optimal protein levels. A complete absence of ELAV causes the activation and nuclear localization of the normally cytoplasmic paralogue FNE, in a process termed EXon-Activated functional Rescue (EXAR). Other species, including mammals, seem to utilize different strategies, such as protein redundancy, to maintain ELAV protein function and effectively safeguard the identity of the neuronal transcriptome. This article is categorized under: RNA Processing > 3' End Processing RNA in Disease and Development > RNA in Development RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

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.

The long noncoding RNA mimi scaffolds neuronal granules to maintain nervous system maturity.

RNA binding proteins and messenger RNAs (mRNAs) assemble into ribonucleoprotein granules that regulate mRNA trafficking, local translation, and turnover. The dysregulation of RNA-protein condensation disturbs synaptic plasticity and neuron survival and has been widely associated with human neurological disease. Neuronal granules are thought to condense around particular proteins that dictate the identity and composition of each granule type. Here, we show in Drosophila that a previously uncharacterized long noncoding RNA, mimi, is required to scaffold large neuronal granules in the adult nervous system. Neuronal ELAV-like proteins directly bind mimi and mediate granule assembly, while Staufen maintains condensate integrity. mimi granules contain mRNAs and proteins involved in synaptic processes; granule loss in mimi mutant flies impairs nervous system maturity and neuropeptide-mediated signaling and causes phenotypes of neurodegeneration. Our work reports an architectural RNA for a neuronal granule and provides a handle to interrogate functions of a condensate independently of those of its constituent proteins.

Repression of the Hox gene abd-A by ELAV-mediated Transcriptional Interference.

Intergenic transcription is a common feature of eukaryotic genomes and performs important and diverse cellular functions. Here, we investigate the iab-8 ncRNA from the Drosophila Bithorax Complex and show that this RNA is able to repress the transcription of genes located at its 3' end by a sequence-independent, transcriptional interference mechanism. Although this RNA is expressed in the early epidermis and CNS, we find that its repressive activity is limited to the CNS, where, in wild-type embryos, it acts on the Hox gene, abd-A, located immediately downstream of it. The CNS specificity is achieved through a 3' extension of the transcript, mediated by the neuronal-specific, RNA-binding protein, ELAV. Loss of ELAV activity eliminates the 3' extension and results in the ectopic activation of abd-A. Thus, a tissue-specific change in the length of a ncRNA is used to generate a precise pattern of gene expression in a higher eukaryote.

ELAV and FNE Determine Neuronal Transcript Signatures through EXon-Activated Rescue.

The production of alternative RNA variants contributes to the tissue-specific regulation of gene expression. In the animal nervous system, a systematic shift toward distal sites of transcription termination produces transcript signatures that are crucial for neuron development and function. Here, we report that, in Drosophila, the highly conserved protein ELAV globally regulates all sites of neuronal 3' end processing and directly binds to proximal polyadenylation sites of target mRNAs in vivo. We uncover an endogenous strategy of functional gene rescue that safeguards neuronal RNA signatures in an ELAV loss-of-function context. When not directly repressed by ELAV, the transcript encoding the ELAV paralog FNE acquires a mini-exon, generating a new protein able to translocate to the nucleus and rescue ELAV-mediated alternative polyadenylation and alternative splicing. We propose that exon-activated functional rescue is a more widespread mechanism that ensures robustness of processes regulated by a hierarchy, rather than redundancy, of effectors.

The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila.

Alternative pre-mRNA splicing (AS) is a critical regulatory mechanism that operates extensively in the nervous system to produce diverse protein isoforms. Fruitless AS isoforms have been shown to influence male courtship behavior, but the underlying mechanisms are unknown. Using genome-wide approaches and quantitative behavioral assays, we show that the P-element somatic inhibitor (PSI) and its interaction with the U1 small nuclear ribonucleoprotein complex (snRNP) control male courtship behavior. PSI mutants lacking the U1 snRNP-interacting domain (PSIΔAB mutant) exhibit extended but futile mating attempts. The PSIΔAB mutant results in significant changes in the AS patterns of ∼1,200 genes in the Drosophila brain, many of which have been implicated in the regulation of male courtship behavior. PSI directly regulates the AS of at least one-third of these transcripts, suggesting that PSI-U1 snRNP interactions coordinate the behavioral network underlying courtship behavior. Importantly, one of these direct targets is fruitless, the master regulator of courtship. Thus, PSI imposes a specific mode of regulatory control within the neuronal circuit controlling courtship, even though it is broadly expressed in the fly nervous system. This study reinforces the importance of AS in the control of gene activity in neurons and integrated neuronal circuits, and provides a surprising link between a pleiotropic pre-mRNA splicing pathway and the precise control of successful male mating behavior.

Alternative polyadenylation coupled to transcription initiation: Insights from ELAV-mediated 3' UTR extension.

Transcription initiation and mRNA maturation were long considered co-occurring but separately regulated events of gene control. In the past decade, gene promoters, the platforms of transcription initiation, have been assigned additional functions such as the regulation of splicing and 3' end processing. In a recent study, Oktaba and Zhang and al. reveal that neural 3' UTR extension is dependent on promoter sequences. In Drosophila neurons, promoter regions of a subset of genes recruit the RNA-binding protein ELAV, which is required for subsequent ELAV-mediated alternative polyadenylation. Intriguingly, RNA Polymerase II pausing at promoters seems to facilitate ELAV recruitment. How transcription initiation and alternative polyadenylation, processes separated by an entire gene length, are functionally linked, remains unsolved. In this article, I summarize recent findings and discuss possible mechanisms.

ELAV links paused Pol II to alternative polyadenylation in the Drosophila nervous system.

Alternative polyadenylation (APA) has been implicated in a variety of developmental and disease processes. A particularly dramatic form of APA occurs in the developing nervous system of flies and mammals, whereby various developmental genes undergo coordinate 3' UTR extension. In Drosophila, the RNA-binding protein ELAV inhibits RNA processing at proximal polyadenylation sites, thereby fostering the formation of exceptionally long 3' UTRs. Here, we present evidence that paused Pol II promotes recruitment of ELAV to extended genes. Replacing promoters of extended genes with heterologous promoters blocks normal 3' extension in the nervous system, while extension-associated promoters can induce 3' extension in ectopic tissues expressing ELAV. Computational analyses suggest that promoter regions of extended genes tend to contain paused Pol II and associated cis-regulatory elements such as GAGA. ChIP-seq assays identify ELAV in the promoter regions of extended genes. Our study provides evidence for a regulatory link between promoter-proximal pausing and APA.

ELAV mediates 3' UTR extension in the Drosophila nervous system.

Post-transcriptional gene regulation is prevalent in the nervous system, where multiple tiers of regulatory complexity contribute to the development and function of highly specialized cell types. Whole-genome studies in Drosophila have identified several hundred genes containing long 3' extensions in neural tissues. We show that ELAV (embryonic-lethal abnormal visual system) is a key mediator of these neural-specific extensions. Misexpression of ELAV results in the ectopic synthesis of long messenger RNAs (mRNAs) in transgenic embryos. RNA immunoprecipitation assays suggest that ELAV directly binds the proximal polyadenylation signals of many target mRNAs. Finally, ELAV is sufficient to suppress 3' end formation at a strong polyadenylation signal when tethered to a synthetic RNA. We propose that this mechanism for coordinating 3' UTR extension may be generally used in a variety of cellular processes.

Neural-specific elongation of 3' UTRs during Drosophila development.

The 3' termini of eukaryotic mRNAs influence transcript stability, translation efficiency, and subcellular localization. Here we report that a subset of developmental regulatory genes, enriched in critical RNA-processing factors, exhibits synchronous lengthening of their 3' UTRs during embryogenesis. The resulting UTRs are up to 20-fold longer than those found on typical Drosophila mRNAs. The large mRNAs emerge shortly after the onset of zygotic transcription, with several of these genes acquiring additional, phased UTR extensions later in embryogenesis. We show that these extended 3' UTR sequences are selectively expressed in neural tissues and contain putative recognition motifs for the translational repressor, Pumilio, which also exhibits the 3' lengthening phenomenon documented in this study. These findings suggest a previously unknown mode of posttranscriptional regulation that may contribute to the complexity of neurogenesis or neural function.

Drosophila microRNAs 263a/b confer robustness during development by protecting nascent sense organs from apoptosis.

miR-263a/b are members of a conserved family of microRNAs that are expressed in peripheral sense organs across the animal kingdom. Here we present evidence that miR-263a and miR-263b play a role in protecting Drosophila mechanosensory bristles from apoptosis by down-regulating the pro-apoptotic gene head involution defective. Both microRNAs are expressed in the bristle progenitors, and despite a difference in their seed sequence, they share this key common target. In miR-263a and miR-263b deletion mutants, loss of bristles appears to be sporadic, suggesting that the role of the microRNAs may be to ensure robustness of the patterning process by promoting survival of these functionally specified cells. In the context of the retina, this mechanism ensures that the interommatidial bristles are protected during the developmentally programmed wave of cell death that prunes excess cells in order to refine the pattern of the pupal retina.

The conserved microRNA miR-8 tunes atrophin levels to prevent neurodegeneration in Drosophila.

microRNAs (miRNAs) bind to specific messenger RNA targets to posttranscriptionally modulate their expression. Understanding the regulatory relationships between miRNAs and targets remains a major challenge. Many miRNAs reduce expression of their targets to inconsequential levels. It has also been proposed that miRNAs might adjust target expression to an optimal level. Here we analyze the consequences of mutating the conserved miRNA miR-8 in Drosophila. We identify atrophin as a direct target of miR-8. miR-8 mutant phenotypes are attributable to elevated atrophin activity, resulting in elevated apoptosis in the brain and in behavioral defects. Reduction of atrophin levels in miR-8-expressing cells to below the level generated by miR-8 regulation is detrimental, providing evidence for a "tuning target" relationship between them. Drosophila atrophin is related to the atrophin family of mammalian transcriptional regulators, implicated in the neurodegenerative disorder DRPLA. The regulatory relationship between miR-8 and atrophin orthologs is conserved in mammals.

A conserved role for cytoplasmic poly(A)-binding protein 1 (PABPC1) in nonsense-mediated mRNA decay.

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs with premature translation termination codons (PTCs). The mechanisms by which PTCs and natural stop codons are discriminated remain unclear. We show that the position of stops relative to the poly(A) tail (and thus of PABPC1) is a critical determinant for PTC definition in Drosophila melanogaster. Indeed, tethering of PABPC1 downstream of a PTC abolishes NMD. Conversely, natural stops trigger NMD when the length of the 3' UTR is increased. However, many endogenous transcripts with exceptionally long 3' UTRs escape NMD, suggesting that the increase in 3' UTR length has co-evolved with the acquisition of features that suppress NMD. We provide evidence for the existence of 3' UTRs conferring immunity to NMD. We also show that PABPC1 binding is sufficient for PTC recognition, regardless of cleavage or polyadenylation. The role of PABPC1 in NMD must go beyond that of providing positional information for PTC definition, because its depletion suppresses NMD under conditions in which translation efficiency is not affected. These findings reveal a conserved role for PABPC1 in mRNA surveillance.

Translation-independent inhibition of mRNA deadenylation during stress in Saccharomyces cerevisiae.

Post-transcriptional control mechanisms play an important role in regulating gene expression during cellular responses to stress. For example, many stresses inhibit translation, and at least some stresses inhibit mRNA turnover in yeast and mammalian cells. We show that hyperosmolarity, heat shock, and glucose deprivation stabilize multiple mRNAs in yeast, primarily through inhibition of deadenylation. Although these stresses inhibit translation and promote the movement of mRNAs into P-bodies, we also observed inhibition of deadenylation in cycloheximide-treated cells as well as in a mutant strain where translation initiation is impaired. This argues that inhibition of poly(A)-shortening is independent of the translational state of the mRNAs and can occur when mRNAs are localized in polysomes or are not engaged in translation. Analysis of pan2Delta or ccr4Delta strains indicates that stress inhibits the function of both the Ccr4p/Pop2p/Notp and the Pan2p/Pan3p deadenylases. We suggest that under stress, simultaneous repression of translation and deadenylation allows cells to selectively translate mRNAs specific to the stress response, while retaining the majority of the cytoplasmic pool of mRNAs for later reuse and recovery from stress. Moreover, because various cellular stresses also inhibit deadenylation in mammalian cells, this mechanism is likely to be a conserved aspect of the stress response.

In vivo analysis of mRNA stability using the Tet-Off system in the chicken embryo.

The rate of mRNA degradation plays an important role in the control of gene expression. The mRNA stability is mainly dependent on cis-regulatory elements contained in the 3' or 5' untranslated region (UTR) of the mature mRNAs, and its regulation is an efficient way to adapt the level of a given transcript in the cell. Although this process has been well studied in cell culture, little is known about mRNA stability during embryonic development. Here, we describe an assay that combines the tetracyclin-dependent inducible system Tet-Off with in ovo electroporation to monitor mRNA stability in the chick neural tube. We show, by using the GFP intensity as an indirect reporter system, that the 3'UTR of Lunatic Fringe strongly destabilizes transcripts, while transcripts bearing the 3'UTR of Fgf8 are much more stable. This simple assay provides a powerful tool to study mRNA dynamics in vivo.