Biology subjects

Bartel, D. P.

Publications and source records attributed to Bartel, D. P..

At least 19 recordsLinked to original sources

bioRxiv2026

The molecular determinants of PABPC-mediated deadenylation rate

Deadenylation, the enzymatic shortening of the poly(A) tail, is typically the first committed step of mRNA decay. Deadenylation rates span nearly a 1000-fold range between transcripts and are governed by protein-RNA interactions, including those involving cytoplasmic poly(A)-binding protein (PABPC). Previous work shows that PABPC can straddle the junction between the poly(A) tail and the 3' untranslated region (UTR), but whether this conformation influences deadenylation has not been tested. To investigate how straddling influences deadenylation kinetics, we designed a library of tailed RNA substrates and measured both in vitro deadenylation rates in the presence of PABPC and PABPC binding propensity for each substrate. We found that 3' UTR sequences influence deadenylation through two mechanisms. First, structured UTRs are deadenylated more slowly in the absence of PABPC1, an effect that is alleviated by PABPC1. Second, sequences upstream of the poly(A) tail modulate PABPC1 binding propensity, with tighter binding correlating with slower deadenylation. This relationship is abolished with a PABPC1 mutant lacking UTR-binding capacity. Together, these results show that sequences upstream of the poly(A) tail tune PABPC1 binding and deadenylation rates, likely contributing to the range of deadenylation rates observed for cellular mRNAs.

biochemistry↗

bioRxiv2026

Dissection of Poly(A)-binding protein (PABPC) cellular function using degron-mediated depletion with replacement

Cytoplasmic poly(A)-binding proteins (PABPCs) are essential and highly abundant regulators of mRNA stability and translation, but their cellular functions have been difficult to dissect due to slow turnover and the lethality observed upon loss of both major paralogs, PABPC1 and PABPC4. To enable structure-function analysis of PABPC in human cells, we developed a protein-replacement platform that couples rapid auxin-inducible degradation of endogenous PABPC1 and PABPC4 with doxycycline-controlled expression of engineered PABPC variants. This approach enables acute removal of native PABPC and real-time assessment of how specific domains, paralogs, and sequence alterations support cellular fitness, shape transcriptome profiles, and regulate poly(A)-tail length. Using this system, we show that the RRM4 domain of PABPC1 is essential for growth, whereas post-translationally modified lysines within RRM4 are individually dispensable. Paralogs and variants with heterologous RRM4 domains vary in their ability to substitute for PABPC1, revealing functional divergence among PABPCs. Transcriptome profiling identifies variant-specific regulatory signatures, and dose-controlled rescue further delineates the relationship between PABPC variant abundance and global poly(A)-tail lengths in vivo. Together, this platform provides a generalizable strategy for dissecting PABPC biology in the cellular context using rationally designed variants.

molecular biology↗

bioRxiv2026

The spliceosome assembles on excised linear introns to protect them from degradation

In Saccharomyces cerevisiae, prolonged cellular stress induces some introns to accumulate post-splicing as stable, linear, spliceosome-protected RNAs1. These stable introns are defined by having short distances from their branchpoint (BP) sequences to their 3'-splice sites (3'SSs). Stable introns sequester splicing components, thereby reducing splicing activity and affecting cell growth in the stressed conditions. The mechanism by which these normally ephemeral products of pre-mRNA splicing persist cannot be explained by the current understanding of the splicing pathway, which derives primarily from studies of unstressed cells and their extracts2,3. Here, we determined the cryo-electron microscopy (cryo-EM) structure of a stable-intron complex purified from saturated-culture conditions. This structure and experimental follow-up show that a Bact-like spliceosome protects stable introns from degradation, and that the short BP-3'SS distances of stable introns render this conformation of the spliceosome resistant to remodelling by helicases. Spliceosomes can also assemble onto artificial introns that have the same sequences as authentic stable introns but do not rely on splicing for their biogenesis, which demonstrates that spliceosomes arrive at this Bact-like conformation by reassembling onto linear introns after their excision from pre-mRNAs. This reassembly activity is maintained in both stressed and unstressed cells. Thus, most yeast introns compete with pre-mRNAs for access to the splicing machinery, and budding yeast has co-opted this activity to adapt to environmental insults.

molecular biology↗

bioRxiv2026

The E3 ubiquitin ligase mechanism specifying target-directed microRNA degradation

MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to form complexes that down-regulate target RNAs, including mRNAs from most human genes1-3. Within each complex, the miRNA pairs to target mRNAs to specify their repression, and AGO provides effector function while also protecting the miRNA from cellular nucleases2-5. Although much has been learned about this mode of posttranscriptional gene regulation, less is known about how the miRNAs themselves are regulated. In one such regulatory pathway, unusual miRNA targets called "trigger" RNAs reverse the canonical regulatory logic and instead down-regulate microRNAs6-21. This target-directed miRNA degradation (TDMD) is thought to require a cullin-RING E3 ligase (CRL) because it depends on the cullin protein CUL3 and other ubiquitylation components, including the BC-box protein ZSWIM8 (ref. 22,23). ZSWIM8 is required for murine perinatal viability and for destabilization of most short-lived miRNAs, but is otherwise poorly understood23-25. Here, we demonstrate that a human AGO-miRNA- trigger complex selectively binds ZSWIM8 for CUL3-mediated polyubiquitylation of the AGO protein within this complex. Cryogenic electron-microscopy (cryo-EM) analyses show how ZSWIM8 recognizes the distinct AGO2 and miRNA-trigger conformations shaped by pairing of the miRNA to the trigger. For example, this pairing extracts the miRNA from a binding pocket within AGO2, allowing the pocket to be captured by ZSWIM8, and it directs the trigger RNA along a distinct trajectory to be also recognized by ZSWIM8. These results biochemically establish AGO binding and polyubiquitylation as the key regulatory step of TDMD, define a unique CRL class, and reveal generalizable RNA-RNA, RNA-protein, and protein-protein interactions that specify the ubiquitin-mediated degradation of AGO with exquisite selectivity. The substrate features recognized by the E3 ubiquitin ligase do not conform to a conventional degron26-28, but rather establish a two-RNA-factor authentication mechanism specifying a protein ubiquitylation substrate.

biochemistry↗

bioRxiv2025

mRNA 3' UTRs direct microRNA degradation to participate in imprinted gene networks and regulate growth

MicroRNAs direct downregulation of target mRNAs. Sometimes, however, this regulatory paradigm inverts, and a target RNA triggers the degradation of a microRNA. This target-directed microRNA degradation (TDMD) requires ZSWIM8. Zswim8-/- mice exhibit reduced growth and perinatal lethality, accompanied by stabilization of dozens of microRNAs. Nonetheless, studies of TDMD function in mammals have been limited because only two TDMD-triggering RNAs have been identified in mice. Here, we computationally identify and validate five new TDMD-triggering sites in mouse models. One site in Atp6v1g1 and two in Lpar4 direct degradation of miR-335-3p, which shows that in mammals, two sites in the same transcript, and multiple sites in different transcripts, can collaborate to destabilize a microRNA. Moreover, sites in Plagl1 and Lrrc58 direct degradation of miR-322 and miR-503, respectively. Mice lacking the Plagl1 and Lrrc58 sites exhibit reduced growth, demonstrating that target-directed degradation of miR-503 and miR-322 promotes mammalian growth. Both miR-335-3p and Plagl1 are maternally imprinted, implying that they participate in parental conflict, but their corresponding triggers or target microRNA partner are not imprinted. Thus, 3' UTRs directly participate in parental conflict by engaging TDMD to access an additional layer of regulation within a network of imprinted and biallelic genes.

molecular biology↗

bioRxiv2025

5' untranslated regions tune Toxoplasma translation

Some of the longest 5' untranslated regions (UTRs) documented in eukaryotes belong to parasites of the phylum Apicomplexa. Translational regulation plays prominent roles in the development of these parasites, including the agents of toxoplasmosis (Toxoplasma gondii) and malaria. To understand the function of 5' UTRs in apicomplexan translation, we performed high-resolution ribosome profiling of T. gondii in human fibroblasts. We show that parasite translation efficiency (TE) is largely controlled by 5' UTR features and tuned by the number of upstream AUGs (uAUGs). Examination of ribosome occupancy reveals that, despite widespread assembly of parasite monosomes on uAUGs, ribosomes seldom translate uORFs. These determinants of translation are reaffirmed in a massively parallel reporter assay examining the effect of more than 30,000 synthetic 5' UTRs in T. gondii. A model trained on these results accurately predicted the TE of newly designed 5' UTRs. Together, this work defines the regulatory language of T. gondii translation, providing a framework to understand the evolution of exceptionally long 5' UTRs in apicomplexans.

molecular biology↗

bioRxiv2025

Derepression of a single microRNA target causes female infertility in mice

The miR-200a and miR-200b families control mouse ovulation and are essential for female fertility. The ZEB1 transcription factor is a conserved target of both families and has been implicated as a key player in female fertility at multiple levels. Using gene-edited mice that express a miR-200a/b-resistant form of Zeb1, we found that derepression of Zeb1 in the female pituitary caused decreased production of luteinizing hormone and anovulatory infertility. These phenotypes were accompanied by widespread changes in pituitary gene expression characterized by decreased levels of ZEB1 targets, which include the miR-200a/b miRNAs, as expected from the miR-200a/b-ZEB1 double-negative feedback loop. Also observed were increased levels of mesenchymal genes, neuronal genes, and miR-200a/b targets. These results show that a double-negative feedback loop centered on the miRNA regulation of a single transcription factor can significantly influence the expression of thousands of genes and have dramatic phenotypic consequences.

molecular biology↗

bioRxiv2025

Functional microRNA targeting without seed pairing

MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to serve as guides, directing binding to partially complementary sites in mRNAs, ultimately causing post-transcriptional repression. Complementarity to the miRNA seed region (miRNA nucleotides 2-7) is typically both necessary and sufficient for repression. Here, we investigate unusual sites with extensive complementarity to the miRNA 3' region (nucleotide 9 and onwards) but without complementarity to the seed. Top 3'-only sites bind as well as top canonical sites and impart similar repression, which can be further boosted by as few as 2-3 additional pairs to the miRNA seed. Despite these similarities, 3'-only sites have slower association and dissociation rates than seed-matched sites. They also impart different conformations to bound AGO-miRNA complexes than do seed-matched sites, and individual miRNAs differ substantially with respect to how well they bind their respective 3'-only sites. Thus, pairing to the seed is not required for binding and repression, or for a target to gain access to the 3' region of the guide. Overall, for those miRNAs which recognize 3'-only sites, those sites are estimated to constitute 0.5-1% of the endogenous targetome, a proportion resembling that of other rare but functional site types, such as 3'-compensatory sites. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/641270v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1e6045corg.highwire.dtl.DTLVardef@2859eborg.highwire.dtl.DTLVardef@12761beorg.highwire.dtl.DTLVardef@820fe6_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

The G3BP Stress-Granule Proteins Reinforce the Translation Program of the Integrated Stress Response

When mammalian cells are exposed to stress, they coordinate the condensation of stress granules (SGs) through the action of proteins G3BP1 and G3BP2 (G3BPs) and, simultaneously, undergo a massive reduction in translation. Although SGs and G3BPs have been linked to this translation response, their overall impact has been unclear. Here, we investigate the question of how, and indeed whether, G3BPs and SGs shape the stress translation response. We find that SGs are enriched for mRNAs that are resistant to the stress-induced translation shutdown. Although the accurate recruitment of these stress-resistant mRNAs does require the context of stress, a combination of optogenetic tools and spike-normalized ribosome profiling demonstrates that G3BPs and SGs are necessary and sufficient to both help prioritize the translation of their enriched mRNAs and help suppress cytosolic translation. Together these results support a model in which G3BPs and SGs reinforce the stress translation program by prioritizing the translation of their resident mRNAs.

molecular biology↗

bioRxiv2024

The structural basis for RNA slicing by human Argonaute2

Argonaute (AGO) proteins associate with guide RNAs to form complexes that slice transcripts that pair to the guide. This slicing drives post-transcriptional gene-silencing pathways that are essential for many eukaryotes and the basis for new clinical therapies. Despite this importance, structural information on eukaryotic AGOs in a fully paired, slicing-competent conformation--hypothesized to be intrinsically unstable--has been lacking. Here we present the cryogenic-electron microscopy structure of a human AGO-guide complex bound to a fully paired target, revealing structural rearrangements that enable this conformation. Critically, the N domain of AGO rotates to allow the RNA full access to the central channel and forms contacts that license rapid slicing. Moreover, a conserved loop in the PIWI domain secures the RNA near the active site to enhance slicing rate and specificity. These results explain how AGO accommodates targets possessing the pairing specificity typically observed in biological and clinical slicing substrates.

molecular biology↗

bioRxiv2024

Widespread destabilization of C. elegans microRNAs by the E3 ubiquitin ligase EBAX-1

MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to form complexes that direct mRNA repression. miRNAs are also the subject of regulation. For example, some miRNAs are destabilized through a pathway in which pairing to specialized transcripts recruits the ZSWIM8 E3 ubiquitin ligase, which polyubiquitinates AGO, leading to its degradation and exposure of the miRNA to cellular nucleases. Here, we found that 22 miRNAs in C. elegans are sensitive to loss of EBAX-1, the ZSWIM8 ortholog in nematodes, implying that these 22 miRNAs might be subject to this pathway of target-directed miRNA degradation (TDMD). The impact of EBAX-1 depended on the developmental stage, with the greatest effect on the miRNA pool (14.5%) observed in L1 larvae and the greatest number of different miRNAs affected (17) observed in germline-depleted adults. The affected miRNAs included the miR-35-42 family, as well as other miRNAs among the least stable in the worm, suggesting that TDMD is a major miRNA-destabilization pathway in the worm. The excess miR-35-42 molecules that accumulated in ebax-1 mutants caused increased repression of their predicted target mRNAs and underwent 3' trimming over time. In general, however, miRNAs sensitive to EBAX-1 loss had no consistent pattern of either trimming or tailing. Replacement of the 3' region of miR-43 substantially reduced EBAX-1 sensitivity, a result that differed from that observed previously for miR-35. Together, these findings broaden the implied biological scope of TDMD-like regulation of miRNA stability in animals, and indicate that a role for miRNA 3' sequences is variable in the worm.

molecular biology↗

bioRxiv2024

Nuclear release of eIF1 globally increases stringency of start-codon selection to preserve mitotic arrest physiology

Regulated start-codon selection has the potential to reshape the proteome through the differential production of uORFs, canonical proteins, and alternative translational isoforms. However, conditions under which start-codon selection is altered remain poorly defined. Here, using transcriptome-wide translation initiation site profiling, we reveal a global increase in the stringency of start-codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This increased stringency of start-codon selection during mitosis results from increased interactions between the key regulator of start-codon selection, eIF1, and the 40S ribosome. We find that increased eIF1-40S ribosome interactions during mitosis are mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the changes to translational stringency during mitosis, resulting in altered mitotic proteome composition. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage following treatment with anti-mitotic chemotherapeutics. Thus, cells globally control translation initiation stringency with critical roles during the mammalian cell cycle to preserve mitotic cell physiology.

molecular biology↗

bioRxiv2023

The guide RNA sequence dictates the slicing kinetics and conformational dynamics of the Argonaute silencing complex

SUMMARYThe RNA-induced silencing complex (RISC), which powers RNA interference (RNAi), consists of a guide RNA and an Argonaute protein that slices target RNAs complementary to the guide. We find that for different guide-RNA sequences, slicing rates of perfectly complementary, bound targets can be surprisingly different (>250-fold range), and that faster slicing confers better knockdown in cells. Nucleotide sequence identities at guide-RNA positions 7, 10, and 17 underlie much of this variation in slicing rates. Analysis of one of these determinants implicates a structural distortion at guide nucleotides 6-7 in promoting slicing. Moreover, slicing directed by different guide sequences has an unanticipated, 600-fold range in 3'-mismatch tolerance, attributable to guides with weak (AU-rich) central pairing requiring extensive 3' complementarity (pairing beyond position 16) to more fully populate the slicing-competent conformation. Together, our analyses identify sequence determinants of RISC activity and provide biochemical and conformational rationale for their action. HIGHLIGHTSO_LISequence of guide RNA can alter slicing rate of fully paired substrate by 250-fold C_LIO_LISequences that cause more rapid slicing direct more efficient RNAi in cells C_LIO_LIStrong central pairing imparts tolerance for mismatches to the guide 3' region C_LIO_LIThis tolerance is attributable to more fully populating the slicing conformation C_LI

biochemistry↗

bioRxiv2022

A statistical approach for identifying primary substrates of ZSWIM8-mediated microRNA degradation in small-RNA sequencing data

BackgroundOne strategy for identifying targets of a regulatory factor is to perturb the factor and use high-throughput RNA sequencing to examine the consequences. However, distinguishing direct targets from secondary effects and experimental noise can be challenging when confounding signal is present in the background at varying levels. ResultsHere, we present a statistical modeling strategy to identify microRNAs (miRNAs) that are primary substrates of target-directed miRNA degradation (TDMD) mediated by ZSWIM8. This method uses a bi-beta-uniform mixture (BBUM) model to separate primary from background signal components, leveraging the expectation that primary signal is restricted to upregulation and not downregulation upon loss of ZSWIM8. The BBUM model strategy retained the apparent sensitivity and specificity of the previous ad hoc approach but was more robust against outliers, achieved a more consistent stringency, and could be performed using a single cutoff of false discovery rate (FDR). ConclusionsWe developed the BBUM model, a robust statistical modeling strategy to account for background secondary signal in differential expression data. It performed well for identifying primary substrates of TDMD and should be useful for other applications in which the primary regulatory targets are only upregulated or only downregulated. The BBUM model, FDR-correction algorithm, and significance-testing methods are available as an R package at https://github.com/wyppeter/bbum.

bioinformatics↗

bioRxiv2021

Pairing to the microRNA 3' region occurs through two alternative binding modes, with affinity shaped by nucleotide identity as well as pairing position

MicroRNAs (miRNAs), in association with Argonaute (AGO) proteins, direct repression by pairing to sites within mRNAs. Compared to pairing preferences of the miRNA seed region (nucleotides 2-8), preferences of the miRNA 3' region are poorly understood, due to the sparsity of measured affinities for the many pairing possibilities. We used RNA bind-n-seq with purified AGO2-miRNA complexes to measure relative affinities of >1,000 3'-pairing architectures for each miRNA. In some cases, optimal 3' pairing increased affinity by >500-fold. Some miRNAs had two high-affinity 3'-pairing modes--one of which included additional nucleotides bridging seed and 3' pairing to enable high-affinity pairing to miRNA nucleotide 11. The affinity of binding and the position of optimal pairing both tracked with the occurrence of G or oligo(G/C) nucleotides within the miRNA. These and other results advance understanding of miRNA targeting, providing insight into how optimal 3' pairing is determined for each miRNA. HIGHLIGHTSO_LIRNA bind-n-seq reveals relative affinities of >1,000 3'-pairing architectures C_LIO_LITwo distinct 3'-binding modes can enhance affinity, by >500-fold in some instances C_LIO_LIG and oligo(G/C) residues help define the miRNA 3' segment most critical for pairing C_LIO_LISeed mismatch identity can influence the contribution of compensatory 3' pairing C_LI

biochemistry↗

bioRxiv2021

The molecular basis of coupling between poly(A)-tail length and translational efficiency

In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail length and TE disappears. Here, we elucidate how this coupling is first established and why it disappears. Overexpressing cytoplasmic poly(A)-binding protein (PABPC) in frog oocytes specifically improved translation of short-tailed mRNAs, thereby diminishing coupling between tail length and TE. Thus, coupling requires limiting PABPC, implying that in coupled systems longer-tail mRNAs better compete for limiting PABPC. In addition to expressing excess PABPC, post-embryonic cells had two other properties that prevented strong coupling: terminal-uridylation-dependent destabilization of mRNAs lacking bound PABPC, and a regulatory regime wherein PABPC contributes minimally to TE. Thus, these results revealed three fundamental mechanistic requirements for coupling and defined the context-dependent functions for PABPC, in which this protein promotes TE but not mRNA stability in coupled systems and mRNA stability but not TE in uncoupled systems.

molecular biology↗

bioRxiv2019

Xrn1p Acts at Multiple Steps in the Budding-Yeast RNAi Pathway to Enhance the Efficiency of Silencing

RNA interference (RNAi) is a gene-silencing pathway that can play roles in viral defense, transposon silencing, heterochromatin formation, and post-transcriptional gene silencing. Although absent from Saccharomyces cerevisiae, RNAi is present in other budding-yeast species, including Naumovozyma castellii, which have an unusual Dicer and a conventional Argonaute that are both required for gene silencing. To identify other factors that act in the budding-yeast pathway, we performed an unbiased genetic selection. This selection identified Xrn1p, the cytoplasmic 5'-to-3' exoribonuclease, as a cofactor of RNAi in budding yeast. Deletion of XRN1 impaired gene silencing in N. castellii, and this impaired silencing was attributable to multiple functions of Xrn1p, including affecting the composition of siRNA species in the cell, influencing the efficiency of siRNA loading into Argonaute, degradation of cleaved passenger strand, and degradation of sliced target RNA.

molecular biology↗

bioRxiv2019

MicroRNAs Cause Accelerated Decay of Short-Tailed Target mRNAs

MicroRNAs (miRNAs) specify recruitment of deadenylases to mRNA targets. Despite this recruitment, we find that miRNAs have almost no effect on steady-state poly(A)-tail lengths of their targets in mouse fibroblasts, which motivates acquisition of pre-steady-state measurements of the effects of miRNAs on tail lengths, mRNA levels, and translational efficiencies. Effects on translational efficiency are minimal compared to effects on mRNA levels--even for newly transcribed target mRNAs. Effects on target mRNA levels accumulate as the mRNA population approaches steady state, whereas effects on tail lengths peak for recently transcribed target mRNAs and then subside. Computational modeling of this phenomenon reveals that miRNAs cause not only accelerated deadenylation of their targets but also accelerated decay of short-tailed target molecules. This unanticipated effect of miRNAs largely prevents short-tailed target mRNAs from accumulating despite accelerated target deadenylation. The net result is a nearly imperceptible change to the steady-state tail-length distribution of targeted mRNAs.\n\nHighlightsO_LImiRNAs cause accelerated decay of short-tailed target molecules\nC_LIO_LIThis accelerated decay largely prevents accumulation of short-tailed target mRNAs\nC_LIO_LImiRNAs are similarly effective on short-lived and long-lived target mRNAs\nC_LIO_LIIn 3T3 cells, miRNA effects on translation are negligible--even for nascent mRNA\nC_LI

genomics↗