Biology subjects

Ambros, V.

Publications and source records attributed to Ambros, V..

13 recordsLinked to original sources

bioRxiv2026

Cold Shock Domain Protein LIN-66 cooperates with microRNA-pathway buffering to safeguard developmental timing

Robust execution of developmental cell fates requires precise spatiotemporal control of the fate-defining regulators. In Caenorhabditis elegans, temporal patterning of larval hypodermal fates is governed by the heterochronic gene regulatory network, in which microRNAs act as major post-transcriptional regulators by silencing temporal transcripts through 3'UTR-dependent repression. Here, we investigate lin-66, which encodes a nematode-specific cold shock domain protein previously implicated in heterochronic regulation and reported to associate with the miRISC effector protein AIN-1. Using targeted domain mutations and genetic analysis, we show that LIN-66 activity in the hypodermal cell-fate patterning requires its cold shock domain. Loss of lin-66 causes persistent expression of LIN-14 and LIN-28, two early temporal regulators in the hypodermal seam cells that are canonical microRNA targets. Analysis indicates that lin-66 function in seam-cell fate patterning does not depend on the native 3'UTR sequences of lin-14 or lin-28, distinguishing its activity from canonical microRNA repression.. Consistent However, consistent with the a broad functional overlap between LIN-66 function and microRNA-mediated regulation, hypodermal lin-66 loss-of-function phenotypes are strongly enhanced by mutations in alg-1 and ain-1/2, which encode components of the microRNA-induced silencing complex. Moreover, loss of lin-66 enhances phenotypes in mutants sensitized for microRNA activity outside the hypodermis. Together, these findings identify LIN-66 as a cold shock domain-dependent post-transcriptional regulator that safeguards developmental timing by limiting persistence of early fate regulators through mechanisms that intersect with, but are partly separable from, canonical 3'UTR-mediated microRNA repression.

developmental biology↗

bioRxiv2025

Multi-dimensional regulation of LIN-28 temporal expression dynamics in the C. elegans heterochronic gene cascade

LIN-28 is an evolutionarily conserved RNA-binding protein that plays critical roles in regulating pluripotency and cell fate determination during animal development. In Caenorhabditis elegans, lin-28 is an integral component of the heterochronic (developmental timing) gene regulatory cascade. Loss-of-function mutations in lin-28 result in precocious cell fate determination during larval development. Previous studies showed that the proper progression of stage specific cell fates during larval development depends on the progressive downregulation of LIN-28, which is negatively regulated by the lin-4 microRNA through complementary sequences located in the lin-28 3 UTR. In this study, we employ CRISPR/Cas9 editing of the endogenous lin-28 locus to demonstrate that the robust developmental downregulation of LIN-28 involves contributions from multiple inputs. These include a convergent action of the let-7 family and lin-4 microRNAs via adjacent complementary sites in the lin-28 3 UTR, in conjunction with the previously described post-translational inhibition of LIN-28 by the lep-5 long non-coding RNA, which all together account for virtually the entirety of LIN-28 repression. Furthermore, through the systematic testing of a series of truncations of the lin-28 3 UTR, we identify three positive regulatory regions that enhance LIN-28 expression, thereby counterbalancing the negative effects of the let-7 and lin-4 microRNAs and the lep-5 long non-coding RNA.

developmental biology↗

bioRxiv2024

Identification and developmental profiling of microRNAs in the acoel worm Hofstenia miamia

The acoel worm Hofstenia miamia (H. miamia) has recently emerged as a model organism for studying whole-body regeneration and embryonic development, providing an opportunity to probe post-transcriptional mechanisms in both processes in the same organism. Here, we establish a resource for studying H. miamia microRNA-mediated gene regulation, a major aspect of post-transcriptional control in animals. We generated a PacBio long-read sequencing-based genome assembly. We developed a stringent microRNA annotation framework for H. miamia using small RNA-sequencing samples spanning key developmental stages. Our analysis uncovered 545 microRNA loci, including 154 highest-confidence loci based on structural features, expression levels, and prediction quality metrics. Comparison of microRNA seed sequences with those in other bilaterian species revealed that H. miamia encodes many known conserved bilaterian microRNA families and that several microRNA families previously reported only in protostomes or deuterostomes likely have ancient bilaterian origins. We profiled and characterized the expression dynamics and strand preference of microRNAs in H. miamia embryonic and post-embryonic development. An intron that is spliced in the primary transcript of co-transcribed let-7 and mir-125 microRNAs. To generate hypotheses for microRNA function, we annotated the 3 UTRs of H. miamia protein-coding genes and performed microRNA target site predictions. Focusing on genes that are known to function in the wound response, posterior patterning, and neural differentiation in H. miamia, we found that these processes may be under substantial microRNA regulation. Notably, we found that microRNAs in MIR-7 and MIR-9 families, which have target sites in the posterior genes fz-1, wnt-3, and sp5 are indeed expressed in the anterior of the animal, consistent with an anterior-biased repressive effect on their corresponding target genes. Our annotation provides a resource for future studies of post-transcriptional regulation of gene expression during development and regeneration.

genomics↗

bioRxiv2023

Modeling neurodevelopmental disorder-associated hAGO1 mutations in C. elegans Argonaute ALG-1

MicroRNAs (miRNA) are endogenous non-coding RNAs important for post-transcriptional regulation of gene expression. miRNAs associate with Argonaute proteins to bind to the 3 UTR of target genes and confer target repression. Recently, multiple de novo coding variants in the human Argonaute gene AGO1 (hAGO1) have been reported to cause a neurodevelopmental disorder (NDD) with intellectual disability (ID). Most of the altered amino acids are conserved between the miRNA-associated Argonautes in H. sapiens and C. elegans, suggesting the hAGO1 mutations could disrupt evolutionarily conserved functions in the miRNA pathway. To investigate how the hAGO1 mutations may affect miRNA biogenesis and/or functions, we genetically modeled four of the hAGO1 de novo variants (referred to as NDD mutations) by introducing the identical mutations to the C. elegans hAGO1 homolog, alg-1. This array of mutations caused distinct effects on C. elegans miRNA functions, miRNA populations, and downstream gene expression, indicative of profound alterations in aspects of miRNA processing and miRISC formation and/or activity. Specifically, we found that the alg-1 NDD mutations cause allele-specific disruptions in mature miRNA profiles both in terms of overall abundances and association with mutant ALG-1. We also observed allele-specific profiles of gene expression with altered translational efficiency and/or mRNA abundance. The sets of perturbed genes include human homologs whose dysfunction is known to cause NDD. We anticipate that these cross-clade genetic studies may advance the understanding of fundamental Argonaute functions and provide insights into the conservation of miRNA-mediated post-transcriptional regulatory mechanisms.

molecular biology↗

bioRxiv2021

Critical contribution of 3' non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA

Base-pairing of the seed region (g2-g8) is essential for microRNA targeting, however, the in vivo function of the 3 non-seed region (g9-g22) are less well understood. Here we report the first systematic investigation of the in vivo roles of 3 non-seed nucleotides in microRNA let-7a, whose entire g9-g22 region is conserved among bilaterians. We found that the 3 non-seed sequence functionally distinguishes let-7a from its family paralogs. The complete pairing of g11-g16 is essential for let-7a to fully repress multiple key targets, including evolutionarily conserved lin-41, daf-12 and hbl-1. Nucleotides at g17-g22 are less critical but may compensate for mismatches in the g11-g16 region. Interestingly, the 3 non-seed pairing of let-7a can be critically required with certain minimal complementarity for sites with perfect seed pairing. These results provide evidence that the specific configurations of both seed and 3 non-seed base-pairing can critically influence microRNA-mediated gene regulation in vivo.

molecular biology↗

bioRxiv2020

A cohort of Caenorhabditis species lacking the highly conserved let-7 microRNA

let-7 is a highly conserved microRNA with critical functions integral to cell fate specification and developmental progression in diverse animals. In Caenorhabditis elegans, let-7 is a component of the heterochronic (developmental timing) gene regulatory network, and loss-of-function mutations of let-7 result in lethality during the larval to adult transition due to misregulation of the conserved let-7 target, lin-41. To date, no bilaterian animal lacking let-7 has been characterized. In this study, we identify a cohort of nematode species within the genus Caenorhabditis, closely related to C. elegans, that lack the let-7 microRNA, owing to absence of the let-7 gene. Using C. sulstoni as a representative let-7-lacking species to characterize normal larval development in the absence of let-7, we demonstrate that, except for the lack of let-7, the heterochronic gene network is otherwise functionally conserved. We also report that species lacking let-7 contain a group of divergent let-7 orthologs -- also known as the let-7-family of microRNAs -- that have apparently assumed the role of targeting the lin-41 mRNA. Summary StatementWe have identified a group of Caenorhabditis species that lack let-7a, an otherwise highly conserved and nearly ubiquitous microRNA that was previously thought to be critical to bilaterian animal development.

genetics↗

bioRxiv2019

Regulation of Nuclear-cytoplasmic Partitioning by the lin-28-lin-46 Pathway Reinforces MicroRNA Repression of HBL-1 to Confer Robust Cell-fate Progression in C. elegans

MicroRNAs target complementary mRNAs for degradation or translational repression, reducing or preventing protein synthesis. In C. elegans, the transcription factor HBL-1 (Hunchback-like 1) promotes early larval (L2) stage cell-fate, and the let-7-family microRNAs temporally down-regulate HBL-1 to enable the L2-to-L3 cell-fate progression. In parallel to let-7-family microRNAs, the conserved RNA binding protein LIN-28 and its downstream gene lin-46, also act upstream of HBL-1 in regulating the L2-to-L3 cell-fate progression. The molecular function of LIN-46, and how the lin-28-lin-46 pathway regulates HBL-1, are not understood. Here, we report that the regulation of HBL-1 by the lin-28-lin-46 pathway is independent of the let-7/lin-4 microRNA complementary sites (LCSs) in the hbl-1 3UTR, and involves a stage-specific post-translational regulation of HBL-1 nuclear accumulation. We find that LIN-46 is necessary and sufficient to prevent nuclear accumulation of HBL-1. Our results illuminate that the robust progression from L2 to L3 cell-fates depends on the combination of two distinct modes of HBL-1 down-regulation: decreased synthesis of HBL-1 via let-7-family microRNA activity, and decreased nuclear accumulation of HBL-1 via action of the lin-28-lin-46 pathway. Like HBL-1, many microRNA targets are transcription factors (TFs); and cooperation between regulation of nuclear accumulation and microRNA-mediated control of synthesis rate may be required to increase the precision of or confer robustness to down-regulation of these microRNA target TFs, which can be critical to achieve the optimal phenotypes.

developmental biology↗

bioRxiv2019

C. elegans LIN-28 controls temporal cell-fate progression by regulating LIN-46 expression via the 5’UTR of lin-46 mRNA

Human Lin28 is a conserved RNA-binding protein that promotes proliferation and pluripotency and can be oncogenic. Lin28 and C. elegans LIN-28 bind to precursor RNAs of the conserved, cellular differentiation-promoting, microRNA let-7 and inhibits production of mature let-7 microRNA. Lin28/LIN-28 also binds to and regulates many mRNAs in various cell types. However, the determinants and consequences of these LIN-28-mRNA interactions are not well understood. Here, we report that LIN-28 in C. elegans represses the expression of LIN-46, a downstream protein in the heterochronic pathway, via the 5 UTR of the lin-46 mRNA. We show that both LIN-28 and the 5UTR of lin-46 are required to prevent LIN-46 expression in the L1 and L2 stages, and that precocious LIN-46 expression is sufficient to skip L2 stage proliferative cell-fates, resulting in heterochronic defects similar to the ones observed in lin-28(0) animals. We propose that the lin-46 5UTR mediates LIN-28 binding to and repression of the lin-46 mRNA. Our results demonstrate that precocious LIN-46 expression alone can account for lin-28(0) phenotypes, demonstrating the biological importance of regulation of individual target mRNAs by LIN-28.

developmental biology↗

bioRxiv2019

The Pseudomonas aeruginosa accessory genome elements, including bacterial immune systems effect virulence towards C. elegans

Multicellular animals and bacteria frequently engage in predator-prey and host-pathogen interactions, such as the well-studied relationship between Pseudomonas aeruginosa and the nematode Caenorhabditis elegans. This study investigates the genomic and genetic basis of bacterial-driven variability in P. aeruginosa virulence towards C. elegans. Natural isolates of P. aeruginosa that exhibit diverse genomes display a broad range of virulence towards C. elegans. Using gene association and genetic analysis, we identified accessory genome elements that correlate with virulence, including both known and novel virulence determinants. Among the novel genes, we found a viral-like mobile element, the teg block, that impairs virulence and whose acquisition is restricted by CRISPR-Cas systems. Further genetic and genomic evidence suggests that spacer-targeted elements preferentially associate with lower virulence and suggest a positive, albeit indirect, role for host CRISPR-Cas systems in the restriction of accessory genome elements that may be detrimental to virulence.

evolutionary biology↗

bioRxiv2019

Environmental pheromone and endocrine signals correct heterochronic developmental phenotypes caused by insufficient expression of let-7 family microRNAs in C. elegans

Adverse environmental conditions can affect rates of animal developmental progression and lead to temporary developmental quiescence (diapause), exemplified by the dauer larva stage of the nematode Caenorhabditis elegans. Remarkably, patterns of cell division and temporal cell fate progression in C. elegans larvae are not affected by changes in developmental trajectory. However, the underlying physiological and gene regulatory mechanisms that ensure robust developmental patterning despite substantial plasticity in developmental progression are largely unknown. Here, we report that diapause-inducing environmental pheromone and endocrine signals correct heterochronic developmental cell lineage defects caused by insufficient expression of let-7 family microRNAs in C. elegans. Two conserved endocrine signaling pathways, DAF-7/TGF-{beta} and DAF-2/Insulin, that confer on the larva diapause/non-diapause alternative developmental trajectories, interact with the nuclear hormone receptor, DAF-12, to initiate and regulate a rewiring of the genetic circuitry controlling temporal cell fates. This rewiring includes: 1) repression of the DAF-12 ligand-activated expression of let-7 family microRNAs, and 2) engagement of a novel ligand-independent DAF-12 activity to downregulate the critical let-7 family target Hunchback-like-1 (HBL-1). This alternative HBL-1 downregulation program is responsible for correcting let-7 family insufficiency phenotypes and it requires the activities of certain heterochronic genes, lin-46, lin-4 and nhl-2, that are previously associated with an altered genetic program in post-diapause animals. Our results show how environmental pheromones and endocrine signaling pathways can coordinately regulate both developmental progression and cell fate transitions in C. elegans larvae under stress, so that the developmental schedule of cell fates remains unaffected by changes in developmental trajectory.

developmental biology↗

bioRxiv2018

Trans-splicing of the C. elegans let-7 primary transcript developmentally regulates let-7 microRNA biogenesis and let-7 family microRNA activity

let-7 is a microRNA whose sequence and roles as a regulator of developmental progression are conserved throughout bilaterians. In most systems, transcription of the let-7 locus occurs relatively early in development, whilst processing of let-7 primary transcript into mature microRNA arises later and is associated with cellular differentiation. In C. elegans and other animals, the RNA binding protein LIN-28 post-transcriptionally inhibits let-7 biogenesis at early developmental stages. The mechanisms by which LIN-28 and other factors developmentally regulate let-7 biogenesis are not fully understood. Nor is it understood how the developmental regulation of let-7 might influence the expression or activities of other microRNAs of the same seed family. Here we show that in C. elegans, the primary let-7 transcript (pri-let-7) is trans-spliced to SL1 splice leader at a position downstream of the let-7 precursor stem-loop, producing a short, polyadenylated downstream mRNA. The trans-splicing event negatively impacts the biogenesis of mature let-7 microRNA in cis, likely by destabilizing the upstream pri-let-7 fragment. Moreover, the trans-spliced downstream mRNA contains complimentary sequences to multiple members of the let-7 seed family (let-7fam), and thereby serves as a sponge to negatively regulate let-7fam function in trans. Thus, this study provides evidence for a mechanism by which splicing of a microRNA primary transcript can negatively regulate said microRNA in cis as well as other microRNAs in trans.\n\nHIGHLIGHTSO_LIThe let-7 primary transcript is trans-spliced to produce an RNA that functions as a sponge that negatively regulates the let-7-family microRNAs.\nC_LIO_LITrans-splicing of this RNA negatively impacts let-7 microRNA biogenesis.\nC_LIO_LILIN-28 regulates this trans-splicing event\nC_LI

molecular biology↗

bioRxiv2018

The C. elegans heterochronic gene lin-28 coordinates the timing of hypodermal and somatic gonadal programs for hermaphrodite reproductive system morphogenesis

C. elegans heterochronic genes determine the timing of expression of specific cell fates in particular stages of developing larva. However, their broader roles in coordinating developmental events across diverse tissues has been less well investigated. Here, we show that loss of lin-28, a central heterochronic regulator of hypodermal development, causes reduced fertility associated with abnormal somatic gonad morphology. In particular, the abnormal spermatheca-uterine valve morphology of lin-28(lf) hermaphrodites trap embryos in the spermatheca, which disrupts ovulation and causes embryonic lethality. The same genes that act downstream of lin-28 in the regulation of hypodermal developmental timing also act downstream of lin-28 in somatic gonad morphogenesis and fertility. Importantly, we find that hypodermal expression, but not somatic gonadal expression, of lin-28 is sufficient for restoring normal somatic gonad morphology in lin-28(lf) mutants. We propose that the abnormal somatic gonad morphogenesis of lin-28(lf) hermaphrodites results from temporal discoordination between the accelerated hypodermal development and normally timed somatic gonad development. Thus, our findings exemplify how a cell-intrinsic developmental timing program can also control cell non-autonomous signaling critical for proper development of other interacting tissues.

developmental biology↗

bioRxiv2016

A microRNA family exerts maternal control on sex determination in C. elegans

Gene expression in early animal embryogenesis is in large part controlled post-transcriptionally. Maternally-contributed microRNAs may therefore play important roles in early development. We have elucidated a major biological role of the nematode mir-35 family of maternally-contributed, essential microRNAs. We show that this microRNA family regulates the sex determination pathway at multiple levels, acting both upstream and downstream of her-1 to prevent aberrantly activated male developmental programs in hermaphrodite embryos. The predicted target genes that act downstream of the mir-35 family in this process, sup-26 and nhl-2, both encode RNA binding proteins, thus delineating a previously unknown post-transcriptional regulatory subnetwork within the well-studied sex determination pathway of C. elegans. Genome-wide profiling of SUP-26 binding targets reveals 775 mRNAs, most of which have no known role in sex determination, suggesting that the mir-35 family may modulate numerous other pathways via regulation of sup-26. Since sex determination in C. elegans requires zygotic gene expression to read the sex chromosome karyotype, early embryos must remain gender-naive; our findings show that the mir-35 family microRNAs act in the early embryo to function as a developmental timer that preserves naivete and prevents premature deleterious developmental decisions.

developmental biology↗