Biology subjects

Smibert, C. A.

Publications and source records attributed to Smibert, C. A..

10 recordsLinked to original sources

bioRxiv2026

Pumilio and Brain tumor regulate the expression of Smaug during the Drosophila maternal-to-zygotic transition

During the Drosophila maternal-to-zygotic transition, maternally deposited RNAs are cleared in a temporally controlled manner. The RNA-binding protein Smaug is a major regulator of maternal mRNA decay and its expression at both the RNA and protein level is limited to a narrow temporal window during the MZT. Here we show that Pumilio RNA-binding protein promotes degradation of smaug mRNA at the end of the MZT through Pumilio-binding cis-elements in the smaug 3 untranslated region, and that disruption of this regulation leads to ectopic Smaug protein expression beyond its normal developmental window. We further find that another RNA-binding protein, Brain tumor, also contributes to repression of ectopic Smaug expression. Transcriptome-wide analyses of embryos lacking Pumilio reveal that it directly regulates hundreds of maternal mRNAs after zygotic genome activation, with many of these Pumilio targets also regulated by Brain tumor. The ectopic Smaug protein that results from loss of either Pumilio or Brain tumor causes widespread downregulation of mRNAs that contain Smaug binding sites. Together, these findings define a post-transcriptional regulatory pathway in which Pumilio and Brain tumor ensure orderly progression of the Drosophila maternal-to-zygotic by clearing smaug mRNA and preventing ectopic Smaug activity.

developmental biology↗

bioRxiv2026

Ataxin-2 regulates the synaptonemal complex to ensure chromosome pairing during female meiosis

Sexual reproduction relies on meiotic recombination and the accurate segregation of homologous chromosomes to generate viable, genetically diverse gametes. While the molecular mechanisms of recombination and chromosome segregation are well studied, the upstream regulatory cues that drive expression of key meiotic genes remain poorly understood, especially in metazoans. Emerging evidence suggests that post-transcriptional regulation plays a central role in initiating and coordinating the meiotic program in both fruit flies and mammals. Here, we identify the RNA-binding protein Ataxin-2 (Atx2) as a crucial regulator of meiosis in Drosophila melanogaster. We show that Atx2 positively regulates meiotic factors, especially components of the synaptonemal complex (SC), a structure essential for pairing, recombination and segregation of homologous chromosomes. In Atx2-depleted germ cells, SC component mRNA and protein levels are markedly reduced, leading to defective SC assembly and maintenance. Consequently, homologous chromosomes fail to pair properly, which is essential to ensure homolog segregation and prevent aneuploidy. These findings uncover Atx2 as a key regulator of the SC and highlight an underappreciated layer of gene regulation essential for accurate meiotic chromosome segregation and fertility.

genetics↗

bioRxiv2025

Long-read proteogenomic atlas of human neuronal differentiation reveals isoform diversity informing neurodevelopmental risk mechanisms

RNA splicing shapes neuronal identity and disease risk, yet current maps lack the developmental resolution and depth to resolve this complexity. Here, we integrate deep long-read RNA sequencing and proteomics in iPSC-derived cortical neurons to generate a high-resolution proteogenomic atlas of human neuron development. We identify 182,371 mRNA isoforms (over half previously unknown) and provide direct peptide evidence for the translation of hundreds of novel protein-coding sequences. Population genetics demonstrates that variants affecting novel exons and splice sites are under negative selection, underscoring the potential significance of these isoforms. During neuronal maturation, we observe that ASD risk genes undergo dynamic isoform switching, including microexon inclusion and intron retention, that remodel key protein domains and regulatory regions. Furthermore, we uncover widespread, long-range coordination between splicing and polyadenylation. Finally, our atlas enables variant reinterpretation in ASD, highlighting the value of an isoform-centric view for interpreting pathogenic variation in neurodevelopment.

bioinformatics↗

bioRxiv2025

The ORB2 RNA-binding protein represses translation of its target transcripts during the Drosophila maternal-to-zygotic transition via its functionally conserved Zinc binding 'ZZ' domain

Post-transcriptional regulation is particularly prominent during the maternal-to-zygotic transition (MZT), a developmental phase during which a large proportion of maternally provided mRNAs is repressed and cleared from metazoan embryos. RNA-binding proteins (RBPs) are key components of the post-transcriptional regulatory machinery. We show that the ORB2 RBP, the Drosophila ortholog of human Cytoplasmic Polyadenylation Element Binding Protein (hCPEB) 2-4 protein subfamily, binds to hundreds of maternally provided, rare-codon-enriched mRNAs in early embryos; that ORB2 targets are translationally repressed and unstable during the MZT; identify a U-rich motif enriched in ORB2 targets 3'UTRs; and show that this motif confers ORB2 binding and repression to a luciferase reporter mRNA in S2 tissue culture cells. When tethered to a luciferase reporter, ORB2 and hCPEB2 (but not ORB and hCPEB1) repress translation; the C-terminal Zinc-binding ( ZZ) domain of ORB2 is necessary and sufficient for repression. ORB2 interacts with a suite of post-transcriptional regulators in early embryos; a subset of these interactions is lost upon deletion of the ZZ domain, notably with the Cup repressive complex. ORB2-targets significantly overlap with those previously identified for the repressive RBP, Smaug (SMG). Analysis of the early embryos translatome in the presence or absence of the endogenous ZZ domain shows that mRNAs bound by ORB2 but not by SMG move onto polysomes upon ZZ domain deletion whereas co-bound transcripts do not, consistent with co-regulation of the latter set of transcripts by both RBPs. Our results assign a function to the ZZ domain and position ORB2 in the post-transcriptional network that regulates maternal transcripts during the Drosophila MZT. ARTICLE SUMMARYWe show that Drosophila ORB2, the ortholog of the human CPEB2 RNA-binding protein, negatively regulates its target mRNAs during the maternal-to-zygotic transition via its C-terminal Zinc-binding ( ZZ) domain.

genetics↗

bioRxiv2024

Post-transcriptional regulatory pre-complex assembly drives timely cell-state transitions during differentiation

Complexes that control mRNA stability and translation promote timely cell-state transitions during differentiation by ensuring appropriate expression patterns of key developmental regulators. The Drosophila RNA-binding protein Brain tumor (Brat) promotes degradation of target transcripts during the maternal-to-zygotic transition in syncytial embryos and in uncommitted intermediate neural progenitors (immature INPs). We identified Ubiquitin-specific protease 5 (Usp5) as a Brat interactor essential for the degradation of Brat target mRNAs in both cell types. Usp5 promotes Brat-dedadenylase pre-complex assembly in mitotic neural stem cells (neuroblasts) by bridging Brat and the scaffolding components of deadenylase complexes lacking their catalytic subunits. The adaptor protein Miranda binds the RNA-binding domain of Brat, limiting its ability to bind target mRNAs in mitotic neuroblasts. Cortical displacement of Miranda activates Brat-mediated mRNA decay in immature INPs. We propose that the assembly of an enzymatically inactive and RNA-binding-deficient pre-complex poises mRNA degradation machineries for rapid activation driving timely developmental transitions.

developmental biology↗

bioRxiv2023

Regulation of the Drosophila transcriptome by Pumilio and CCR4-NOT deadenylase

The sequence-specific RNA-binding protein Pumilio controls development of Drosophila; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we utilize knockdown and knockout approaches coupled with RNA-Seq to measure the impact of Pumilio on the transcriptome of Drosophila cells. We also used an improved RNA co-immunoprecipitation method to identify Pumilio bound mRNAs in Drosophila embryos. Integration of these datasets with the content of Pumilio binding motifs across the transcriptome revealed novel direct Pumilio target genes involved in neural, muscle, wing, and germ cell development, and cellular proliferation. These genes include components of Wnt, TGF-beta, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. Additionally, we identified the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pumilio-mediated repression, and observed concordant regulation of Pumilio:CCR4-NOT target mRNAs. Computational modeling revealed that Pumilio binding, binding site number, density, and sequence context are important determinants of regulation. Moreover, the content of optimal synonymous codons in target mRNAs exhibits a striking functional relationship to Pumilio and CCR4-NOT regulation, indicating that the inherent translation efficiency and stability of the mRNA modulates their response to these trans-acting regulatory factors. Together, the results of this work provide new insights into the Pumilio regulatory network and mechanisms, and the parameters that influence the efficacy of Pumilio-mediated regulation.

molecular biology↗

bioRxiv2023

BrainRBPedia: a resource for RNA-binding proteins relevant to neurodevelopmental disorders

RNA-binding proteins (RBPs) are crucial players in the post-transcriptional regulation of mRNA and play major roles in ensuring proper neuronal development and function. Deficits in RBP function have been implicated in a number of neurodevelopmental disorders including autism spectrum disorder (ASD) and intellectual disability (ID), yet we lack resources that integrate current knowledge of RBP function, tissue expression, and disease association in one place to aid in their experimental characterization. Here we introduce BrainRBPedia - a database of 1072 RBPs with both disease annotations for neurodevelopmental disorders and functional annotations relevant to these disorders, including loss-of-function intolerance and expression specificity to the brain, neurons, and neuronal development. Using these functional annotations, we develop a machine learning model to prioritize RBPs likely to be involved in ASD and ID. Our model indicates that RBPs with high loss-of-function intolerance and those upregulated during neuronal differentiation are disproportionately likely to contribute to ASD and ID etiology. In summary, BrainRBPedia comprises a unique resource for researchers interested in the experimental characterization of RBPs in relation to neurodevelopmental disorders and suggests functional signatures of RBPs likely to play a role in neurodevelopment.

bioinformatics↗

bioRxiv2023

Smaug regulates germ plasm synthesis and primordial germ cell number in Drosophila embryos by repressing the oskar and bruno 1 mRNAs

During Drosophila oogenesis, the Oskar (OSK) RNA-binding protein (RBP) determines the amount of germ plasm that assembles at the posterior pole of the oocyte. Here we identify the mechanisms that regulate the osk mRNA in the early embryo. We show that the Smaug (SMG) RBP is transported into the germ plasm of the early embryo where it accumulates in the germ granules. SMG binds to and represses translation of the osk mRNA itself as well as the bruno 1 (bru1) mRNA, which encodes an RBP that we show promotes germ plasm production. Loss of SMG or mutation of SMGs binding sites in the osk or bru1 mRNAs results in ectopic translation of these transcripts in the germ plasm and excess PGCs. SMG therefore triggers a post-transcriptional regulatory pathway that attenuates germ plasm synthesis in embryos, thus modulating the number of PGCs.

developmental biology↗

bioRxiv2020

The RNA-binding protein, Rasputin/G3BP, enhances the stability and translation of its target mRNAs

G3BP RNA-binding proteins are important components of stress granules (SGs). Here we analyze the role of Drosophila G3BP, Rasputin (RIN), in unstressed cells, where RIN is not SG associated. Immunoprecipitation followed by microarray analysis identified over 550 mRNAs that copurify with RIN. The mRNAs found in SGs are long and translationally silent. In contrast, we find that RIN-bound mRNAs, which encode core components of the transcription, splicing and translation machinery, are short, stable and highly translated. We show that RIN is associated with polysomes and provide evidence for a direct role for RIN and its human homologs in stabilizing and upregulating the translation of their target mRNAs. We propose that when cells are stressed the resulting incorporation of RIN/G3BPs into SGs sequesters them away from their short target mRNAs. This would downregulate the expression of these transcripts, even though they are not incorporated into stress granules.

genomics↗

bioRxiv2019

Precise temporal regulation of post-transcriptional repressors is required for an orderly Drosophila maternal-to-zygotic transition

In animal embryos the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. We show that the maternal proteome represents over half of the protein coding capacity of the Drosophila melanogaster genome and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). While the ubiquitin-proteasome system is necessary for clearance of all four repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL and ME31B for degradation early in the MZT; the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG makes the protein immune to degradation. We show that artificially persistent SMG downregulates the zygotic re-expression of mRNAs whose maternal contribution is cleared by SMG. Thus, clearance of SMG permits an orderly MZT.

developmental biology↗