Biology subjects

Taliaferro, M.

Publications and source records attributed to Taliaferro, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Widespread exon definition is promoted by the U1 snRNP 70K subunit and requires sites of contact with RNA Polymerase II

Much pre-mRNA splicing initiates co-transcriptionally, but its mechanism is unclear. We investigated this process by degron depletion of the U1-70K subunit of U1 snRNP that contacts transcribing RNA polymerase II (RNAPII). U1-70K deficiency caused extensive exon skipping consistent with widespread exon definition. Surprisingly, exons with the strongest 5' splice sites (SSs) are skipped. Furthermore, strengthening of non-canonical 5' splice sites with a complementary mutant U1 snRNA or a splice modifying drug induced exon skipping in response to U1-70K depletion. We suggest that U1-70K plays a previously unappreciated role in destabilizing the U1-5'SS interaction to facilitate the transition to the U6-5'SS interaction required for productive splicing following exon definition. Consistent with this idea, knock- down of Prp28/DDX23 that exchanges U1 for U6 at the 5'SS also promotes exon skipping mimicking U1-70K depletion. Replacing WT U1-70K in degron-containing cells with a mutant in the RNAPII interface caused widespread exon skipping. We propose that frequent exon definition occurs at the transcription elongation complex and is enabled by U1-70K contact with transcribing RNAPII.

molecular biology↗

bioRxiv2026

NET1 mRNA localization to the midbody is required for ARP2/3-dependent initiation of mitotic abscission

The recruitment and activation of abscission machinery following mitosis is tightly regulated in time and space, yet the mechanisms controlling this process are poorly understood. We find that RNA localization and local translation at the midbody regulates when and where abscission-regulating proteins are expressed. The 3'UTR of NET1 mRNA contains an element that is necessary and sufficient for RNA targeting to the midbody. Mislocalization of NET1 mRNA results in a loss of NET1 protein, a Rho family GEF, throughout the intercellular bridge as well as slower cell proliferation and delayed abscission. This leads to a loss of Arp2/3 at the abscission site and is dependent upon NET1 binding to Rho family GTPases. These findings establish midbody RNA localization and local translation as a key layer of regulation over abscission timing and identify a role for NET1 as a regulator of Arp2/3-mediated branched actin accumulation at the abscission site.

molecular biology↗

bioRxiv2026

Robust mammalian RNA localization elements are complex and multipartite

The subcellular localization patterns of RNAs are controlled by regulatory elements contained within them. However, for most localized RNAs, the identities of these elements remain unknown. We had previously identified several localization elements that are necessary and sufficient for robust, kinesin-dependent RNA targeting to microtubule plus ends in a variety of cell types. Yet the characteristics of these elements that are critical for function remained unclear. To address this, we systematically created tens of thousands of mutant localization elements and quantified their ability to regulate subcellular RNA localization in neuronal cells. We found that the minimally active size of these localization elements is large, approximately 200 nucleotides. These elements contain multiple important subsequences, with some being completely intolerant of any changes and others being tolerant to a shuffling of nucleotide order but not to changes in nucleotide composition. Using single molecule microscopy, we verified these findings in primary rat neurons. Together, these results demonstrate that highly active mammalian RNA localization elements are large, complex, and multipartite and lay a foundation for further mechanistic studies of their function.

molecular biology↗

bioRxiv2025

TDP-43 directly inhibits RNA accumulation in neurites through modulation of RNA stability

The subcellular localization of hundreds of RNAs to neuronal projections allows neurons to efficiently and rapidly react to spatially restricted external cues. However, for the vast majority of these RNAs, the mechanisms that govern their localization are unknown. Here we demonstrate that the ALS-associated RNA binding protein TDP-43 primarily acts to keep RNAs out of neuronal projections. Using subcellular fractionation and single molecule RNA FISH we find that TDP-43 loss results in the increased neurite accumulation of hundreds of RNAs. These RNAs are highly enriched for known TDP-43 binding sites, suggesting that TDP-43 directly binds them to regulate their localization. We then identified precise regions within RNAs that mediate their TDP-43-dependent localization and interaction with TDP-43 using high-throughput functional assays in cells and high-throughput binding assays in vitro. We found that these regions also mediated TDP-43-dependent RNA instability, identifying the mechanism by which TDP-43 regulates RNA localization. ALS-associated mutations in TDP-43 resulted in similar RNA mislocalization phenotypes as did TDP-43 loss in human iPS-derived motor neurons. These findings establish TDP-43 as a direct negative regulator of RNA abundance in neurites and suggest that mislocalization of specific transcripts may occur in ALS patients.

molecular biology↗

bioRxiv2024

Quantification of subcellular RNA localization through direct detection of RNA oxidation

Across cell types and organisms, thousands of RNAs display asymmetric subcellular distributions. The study of this process often requires quantifying abundances of specific RNAs at precise subcellular locations. To analyze subcellular transcriptomes, multiple proximity-based techniques have been developed in which RNAs near a localized bait protein are specifically labeled, facilitating their biotinylation and purification. However, these complex methods are often laborious and require expensive enrichment reagents. To streamline the analysis of localized RNA populations, we developed Oxidation-Induced Nucleotide Conversion sequencing (OINC-seq). In OINC-seq, RNAs near a genetically encoded, localized bait protein are specifically oxidized in a photo-controllable manner. These oxidation events are then directly detected and quantified using high-throughput sequencing and our software package, PIGPEN, without the need for biotin-mediated enrichment. We demonstrate that OINC-seq can induce and quantify RNA oxidation with high specificity in a dose- and light-dependent manner. We further show the spatial specificity of OINC-seq by using it to quantify subcellular transcriptomes associated with the cytoplasm, ER, nucleus, and the inner and outer membranes of mitochondria. Finally, using transgenic zebrafish, we demonstrate that OINC-seq allows proximity-mediated RNA labeling in live animals. In sum, OINC-seq together with PIGPEN provide an accessible workflow for the analysis of localized RNAs across different biological systems.

molecular biology↗

bioRxiv2024

Trafficking and translation of mRNA in osteocyte dendrites

Osteocyte dendrites extend great distances through canaliculi in bone supported by an extensive cytoskeletal network. The formation and maintenance of osteocyte dendrites is closely linked to bone health, yet the mechanisms regulating these processes are poorly understood. Here we tested the hypothesis that osteocytes transport mRNA to their dendrites for local translation as a mechanism of regulating the formation and maintenance of these subcellular structures. Using molecular and imaging approaches, we demonstrated that a subset of mRNAs are enriched in Ocy454 osteocyte-like cell dendrites. To understand how certain mRNAs are directed to dendrites, we performed a massively parallel reporter assay with 7115 oligonucleotide sequences designed from the 3 untranslated regions of eight dendrite-enriched transcripts. We identified localization sequences within some 3UTRs that were both sufficient and necessary for trafficking to osteocyte dendrites, indicating that localization directions are specifically encoded in enriched transcripts. Furthermore, we found that dendrites contain the elements necessary for local translation, and trafficked transcripts were engaged in active translation within dendrites. Together this work describes a molecular regulatory mechanism novel to osteocyte biology with many potential consequences for the spatiotemporal control of the cytoskeleton and dendrites.

molecular biology↗

bioRxiv2024

The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites

RNA molecules are localized to subcellular regions through interactions between localization-regulatory cis-elements and trans-acting RNA binding proteins (RBPs). However, the identities of RNAs whose localization is regulated by a specific RBP as well as the impacts of that RNA localization on cell function have generally remained unknown. Here, we demonstrate that the RBP HNRNPA2B1 acts to keep specific RNAs out of neuronal projections. Using subcellular fractionation, high-throughput sequencing, and single molecule RNA FISH, we find that hundreds of RNAs demonstrate markedly increased abundance in neurites in HNRNPA2B1 knockout cells. These RNAs often encode motor proteins and are enriched for known HNRNPA2B1 binding sites and motifs in their 3' UTRs. The speed and processivity of microtubule-based transport is impaired in these cells, specifically in their neurites. HNRNPA2B1 point mutations that increase its cytoplasmic abundance relative to wildtype lead to stronger suppression of RNA mislocalization defects than seen with wildtype HNRNPA2B1. We further find that the subcellular localizations of HNRNPA2B1 target RNAs are sensitive to perturbations of RNA decay machinery, suggesting that it is HNRNPA2B1s known role in regulating RNA stability that may explain these observations. These findings establish HNRNPA2B1 as a negative regulator of neurite RNA abundance and link the subcellular activities of motor proteins with the subcellular abundance of the RNAs that encode them.

molecular biology↗

bioRxiv2024

The Unkempt RNA binding protein reveals a local translation program in centriole overduplication

Excess centrosomes cause defects in mitosis, cell-signaling, and cell migration, and therefore their assembly is tightly regulated. Plk4 controls centriole duplication at the heart of centrosome assembly, and elevation of Plk4 promotes centrosome amplification (CA), a founding event of tumorigenesis. Here, we investigate the transcriptional consequences of elevated Plk4 and find Unkempt, a gene encoding an RNA binding protein with roles in translational regulation, to be one of only two upregulated mRNAs. Unk protein localizes to centrosomes and Cep131-positive centriolar satellites and is required for Plk4-induced centriole overduplication in an RNA-binding dependent manner. Translation is enriched at centrosomes and centriolar satellites with Unk and Cep131 promoting this localized translation. A transient centrosomal downregulation of translation occurs early in Plk4-induced CA. CNOT9, an Unk interactor and component of the translational inhibitory CCR4-NOT complex, localizes to centrosomes at this time. In summary, centriolar satellites and Unk promote local translation as part of a translational program that ensures centriole duplication.

cell biology↗

bioRxiv2024

Linguistic inputs must be syntactically parsable to fully engage the language network

Human language comprehension is remarkably robust to ill-formed inputs (e.g., word transpositions). This robustness has led some to argue that syntactic parsing is largely an illusion, and that incremental comprehension is more heuristic, shallow, and semantics-based than is often assumed. However, the available data are also consistent with the possibility that humans always perform rule-like symbolic parsing and simply deploy error correction mechanisms to reconstruct ill-formed inputs when needed. We put these hypotheses to a new stringent test by examining brain responses to a) stimuli that should pose a challenge for syntactic reconstruction but allow for complex meanings to be built within local contexts through associative/shallow processing (sentences presented in a backward word order), and b) grammatically well-formed but semantically implausible sentences that should impede semantics-based heuristic processing. Using a novel behavioral syntactic reconstruction paradigm, we demonstrate that backward- presented sentences indeed impede the recovery of grammatical structure during incremental comprehension. Critically, these backward-presented stimuli elicit a relatively low response in the language areas, as measured with fMRI. In contrast, semantically implausible but grammatically well-formed sentences elicit a response in the language areas similar in magnitude to naturalistic (plausible) sentences. In other words, the ability to build syntactic structures during incremental language processing is both necessary and sufficient to fully engage the language network. Taken together, these results provide strongest to date support for a generalized reliance of human language comprehension on syntactic parsing. Significance statementWhether language comprehension relies predominantly on structural (syntactic) cues or meaning- related (semantic) cues remains debated. We shed new light on this question by examining the language brain areas responses to stimuli where syntactic and semantic cues are pitted against each other, using fMRI. We find that the language areas respond weakly to stimuli that allow for local semantic composition but cannot be parsed syntactically--as confirmed in a novel behavioral paradigm--and they respond strongly to grammatical but semantically implausible sentences, like the famous Colorless green ideas sleep furiously sentence. These findings challenge accounts of language processing that suggest that syntactic parsing can be foregone in favor of shallow semantic processing.

neuroscience↗

bioRxiv2023

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast

The recognition of 5 splice site (5 ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5 ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5 ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5 ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in cold-sensitive growth phenotype and moderate splicing defects. Next, we added an auxin-inducible degron to yLuc7 protein and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondria function in yeast.

biochemistry↗

bioRxiv2023

Constructed languages are processed by the same brain mechanisms as natural languages

What constitutes a language? Natural languages share features with other domains: from math, to music, to gesture. However, the brain mechanisms that process linguistic input are highly specialized, showing little response to diverse non-linguistic tasks. Here, we examine constructed languages (conlangs) to ask whether they draw on the same neural mechanisms as natural languages, or whether they instead pattern with domains like math and programming languages. Using individual-subject fMRI analyses, we show that understanding conlangs recruits the same brain areas as natural language comprehension. This result holds for Esperanto (n=19 speakers) and four fictional conlangs (Klingon (n=10), Navi (n=9), High Valyrian (n=3), and Dothraki (n=3)). These findings suggest that conlangs and natural languages share critical features that allow them to draw on the same representations and computations, implemented in the left-lateralized network of brain areas. The features of conlangs that differentiate them from natural languages--including recent creation by a single individual, often for an esoteric purpose, the small number of speakers, and the fact that these languages are typically learned in adulthood-- appear to not be consequential for the reliance on the same cognitive and neural mechanisms. We argue that the critical shared feature of conlangs and natural languages is that they are symbolic systems capable of expressing an open-ended range of meanings about our outer and inner worlds. Significance StatementWhat constitutes a language has been of interest to diverse disciplines - from philosophy and linguistics to psychology, anthropology, and sociology. An empirical approach is to test whether the system in question recruits the brain system that processes natural languages. In spite of their similarity to natural languages, math and programming languages recruit a distinct brain system. Using fMRI, we test brain responses to stimuli not previously investigated--constructed languages (conlangs)--and find that they are processed by the same brain network as natural languages. Thus, an ability for a symbolic system to express diverse meanings about the world-- but not the recency, manner, and purpose of its creation, or a large user base--is a defining characteristic of a language.

neuroscience↗

bioRxiv2023

Driving and suppressing the human language network using large language models

Transformer models such as GPT generate human-like language and are highly predictive of human brain responses to language. Here, using fMRI-measured brain responses to 1,000 diverse sentences, we first show that a GPT-based encoding model can predict the magnitude of brain response associated with each sentence. Then, we use the model to identify new sentences that are predicted to drive or suppress responses in the human language network. We show that these model-selected novel sentences indeed strongly drive and suppress activity of human language areas in new individuals. A systematic analysis of the model-selected sentences reveals that surprisal and well-formedness of linguistic input are key determinants of response strength in the language network. These results establish the ability of neural network models to not only mimic human language but also noninvasively control neural activity in higher-level cortical areas, like the language network.

neuroscience↗

bioRxiv2023

Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

The cytoplasmic dynein-1 (dynein) motor plays a key role in cellular organisation by transporting a wide variety of cellular constituents towards the minus ends of microtubules. However, relatively little is known about how the biosynthesis, assembly and functional diversity of the motor is orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a guide RNA library targeting 18,253 genes, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints generated from multiplexed images revealed co-functional genes involved in many cellular processes, including several candidate novel regulators of core dynein functions. Mechanistic analysis of one of these proteins, the RNA-binding protein SUGP1, provides evidence that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our dataset represents a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organisation that were captured by our high-content imaging.

cell biology↗

bioRxiv2023

Functional characterization of the language network of polyglots and hyperpolyglots with precision fMRI

How do polyglots--individuals who speak five or more languages--process their languages, and what can this population tell us about the language system? Using fMRI, we identified the language network in each of 34 polyglots (including 16 hyperpolyglots with knowledge of 10+ languages) and examined its response to the native language, non-native languages of varying proficiency, and unfamiliar languages. All language conditions engaged all areas of the language network relative to a control condition. Languages that participants rated as higher-proficiency elicited stronger responses, except for the native language, which elicited a similar or lower response than a non-native language of similar proficiency. Furthermore, unfamiliar languages that were typologically related to the participants high-to-moderate-proficiency languages elicited a stronger response than unfamiliar unrelated languages. The results suggest that the language networks response magnitude scales with the degree of engagement of linguistic computations (e.g., related to lexical access and syntactic-structure building). We also replicated a prior finding of weaker responses to native language in polyglots than non-polyglot bilinguals. These results contribute to our understanding of how multiple languages co-exist within a single brain and provide new evidence that the language network responds more strongly to stimuli that more fully engage linguistic computations.

neuroscience↗

bioRxiv2022

The Novel Role of Midbody-Associated mRNAs in Regulating Abscission

Midbodies (MBs) have been shown to function during telophase as a recruiting hub, especially for ESCRT-III complex subunits, to regulate the abscission step of cytokinesis. However, the molecular machinery governing specific protein targeting and activation at the MB remains poorly understood. Until recently, it was thought that abscission regulating proteins, such as ESCRT-III complex subunits, accumulate at the MB by directly or indirectly binding to the MB resident protein, CEP55. However, recent studies have shown that depletion of CEP55 does not fully block ESCRT-III targeting to the MB, and cells in CEP55 knock-out mice divide normally. Additionally, since MBs are microtubule-rich, proteinaceous structures, it is conceptually hard to imagine how large protein complexes, such as the ESCRT-III complex, can successfully diffuse into the MB from the cytosol in a rapid and highly regulated manner. Here, we show that MBs contain mRNAs and that these MB-associated mRNAs can be locally translated, resulting in the accumulation of abscission-regulating proteins. We also demonstrate that localized MB-associated translation of CHMP4B is required for its targeting to the abscission site and that 3' UTR-dependent CHMP4B mRNA targeting to the MB is required for successful completion of cytokinesis. Finally, we identify regulatory cis-elements within RNAs that are necessary and sufficient for mRNA trafficking to the MB. Based on all this data, we propose a novel method of regulating cytokinesis and abscission by MB-associated targeting and localized translation of selective mRNAs.

cell biology↗

bioRxiv2022

RNA localization mechanisms transcend cell morphology

RNA molecules are localized to specific subcellular regions through interactions between RNA regulatory elements and RNA binding proteins (RBPs). Generally, our knowledge of the mechanistic details behind the localization of a given RNA is restricted to a particular cell type. Here, we show that RNA/RBP interactions that regulate RNA localization in one cell type predictably regulate localization in other cell types with vastly different morphologies. To determine transcriptome-wide RNA spatial distributions across the apicobasal axis of human intestinal epithelial cells, we used our recently developed RNA proximity labeling technique, Halo-seq. We found that mRNAs encoding ribosomal proteins (RP mRNAs) were strongly localized to the basal pole of these cells. Using reporter transcripts and single molecule RNA FISH, we found that pyrimidine-rich TOP motifs in the 5' UTRs of RP mRNAs were su[ffi]cient to drive basal RNA localization. Interestingly, the same TOP motifs were also su[ffi]cient to drive RNA localization to the neurites of mouse neuronal cells. In both cell types, the regulatory activity of the TOP motif was dependent on it being at the extreme 5' end of the transcript, was abolished upon perturbation of the TOP-binding protein LARP1, and was reduced upon inhibition of kinesins. To extend these findings, we compared subcellular RNAseq data from neuronal and epithelial cells. We found that the basal compartment of epithelial cells and the projections of neuronal cells were enriched for highly similar sets of RNAs, indicating that broadly similar mechanisms may be transporting RNAs in both cell types. These findings identify the first RNA element known to regulate RNA localization across the apicobasal axis of epithelial cells, establish LARP1 as an RNA localization regulator, and demonstrate that RNA localization mechanisms cut across cell morphologies.

molecular biology↗

bioRxiv2021

High-throughput identification of RNA localization elements reveals a regulatory role for A/G rich sequences

Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequence elements within them that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we designed and deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous mouse 3' UTRs. We identified peaks of regulatory activity within several 3' UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites in mouse cells as well as active in human neurons. The active localization elements were enriched in adenosine and guanosine residues and were tens to hundreds of nucleotides long as shortening of the elements led to significantly reduced activity. Using RNA affinity and mass spectrometry, we found that the RNA-binding protein Unk was associated with the active localization elements. Depletion of Unk in cells reduced the neurite-enrichment of the elements, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization.

molecular biology↗

bioRxiv2021

Analysis of subcellular transcriptomes by RNA proximity labeling with Halo-seq

Thousands of RNA species display nonuniform distribution within cells. However, quantification of the spatial patterns adopted by individual RNAs remains difficult, in part by a lack of quantitative tools for subcellular transcriptome analysis. In this study, we describe an RNA proximity labeling method that facilitates the quantification of subcellular RNA populations with high spatial specificity. This method, termed Halo-seq, pairs a light-activatable, radical generating small molecule with highly efficient Click chemistry to efficiently label and purify spatially defined RNA samples. We compared Halo-seq with previously reported similar methods and found that Halo-seq displayed a higher efficiency of RNA labeling, indicating that it is well suited to the investigation of small, precisely localized RNA populations. We then used Halo-seq to quantify nuclear, nucleolar, and cytoplasmic transcriptomes, characterize their dynamic nature following perturbation, and identify RNA sequence features associated with their composition. Specifically, we found that RNAs containing AU-rich elements are relatively enriched in the nucleus. This enrichment becomes stronger upon treatment with the nuclear export inhibitor leptomycin B, both expanding the role of HuR in RNA export and generating a comprehensive set of transcripts whose export from the nucleus depends on HuR.

molecular biology↗