Biology subjects

Ferguson, L.

Publications and source records attributed to Ferguson, L..

31 records · Page 2Linked to original sources

bioRxiv2023

eIF3 engages with 3'-UTR termini of highly translated mRNAs in neural progenitor cells

Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3 untranslated region (3-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling, but not with translational efficiency. The results presented here show that eIF3 engages with 3-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.

molecular biology↗

bioRxiv2023

Streamlined and sensitive mono- and diribosome profiling in yeast and human cells

Ribosome profiling has unveiled diverse regulations and perturbations of translation through a transcriptome-wide survey of ribosome occupancy, read out by sequencing of ribosome-protected mRNA fragments. Generation of ribosome footprints and their conversion into sequencing libraries is technically demanding and sensitive to biases that distort the representation of physiological ribosome occupancy. We address these challenges by producing ribosome footprints with P1 nuclease rather than RNase I and replacing RNA ligation with Ordered Two-Template Relay, a single-tube protocol for sequencing library preparation that incorporates adapters by reverse transcription. Our streamlined approach reduced sequence bias and enhanced enrichment of ribosome footprints relative to ribosomal RNA. Furthermore, P1 nuclease preserved a myriad of distinct juxtaposed ribosome complexes informative about yeast and human ribosome fates during translation initiation, stalling, and termination. Our optimized methods for mRNA footprint generation and capture provides a richer translatome profile using lower input and fewer technical challenges.

molecular biology↗

bioRxiv2023

Ribosome rescue factor PELOTA modulates translation start site choice and protein isoform levels of transcription factor C/EBPα

Translation initiation at alternative start sites can dynamically control the synthesis of two or more functionally distinct protein isoforms from a single mRNA. Alternate isoforms of the hematopoietic transcription factor CCAAT-enhancer binding protein (C/EBP) produced from different start sites exert opposing effects during myeloid cell development. This alternative initiation depends on sequence features of the CEBPA transcript, including a regulatory upstream open reading frame (uORF), but the molecular basis is not fully understood. Here we identify trans-acting factors that affect C/EBP isoform choice using a sensitive and quantitative two-color fluorescence reporter coupled with CRISPRi screening. Our screen uncovered a role for the ribosome rescue factor PELOTA (PELO) in promoting expression of the longer C/EBP isoform, by directly removing inhibitory unrecycled ribosomes and through indirect effects mediated by the mechanistic target of rapamycin (mTOR) kinase. Our work provides further mechanistic insights into coupling between ribosome recycling and translation reinitiation in regulation of a key transcription factor, with implications for normal hematopoiesis and leukemiagenesis.

molecular biology↗

bioRxiv2022

Adult ticks (Ixodes scapularis) infected with B. burgdorferi) have increased overwintering survival, with implications for tick population growth

The tick, Ixodes scapularis, vectors pathogens such as Borrelia burgdorferi, the bacterium that causes Lyme Disease. Over the last few decades I. scapularis has expanded its range, introducing a novel health threat into these areas. Warming temperatures appear to be one cause of its range expansion to the north. However, other factors are also involved. We show that unfed adult female ticks infected with B. burgdorferi have greater overwintering survival than uninfected female ticks. Locally collected adult female ticks were placed in individual microcosms and allowed to overwinter in both forest and dune grass environments. In the spring we collected the ticks and tested both dead and living ticks for B. burgdorferi DNA. Infected ticks had greater overwintering survival compared with uninfected ticks every winter for three consecutive winters in both forest and dune grass environments. We discuss the most plausible explanations for this result. The increased winter survival of adult female ticks could enhance tick population growth. Our results suggest that, in addition to climate change, B. burgdorferi infection itself may be promoting the northern range expansion of I. scapularis. Our study highlights how pathogens could work synergistically with climate change to promote host range expansion.

zoology↗

bioRxiv2022

CysPresso: Prediction of cysteine-dense peptide expression in mammalian cells using deep learning protein representations

Background: Cysteine-dense peptides (CDPs) are an attractive pharmaceutical scaffold that display extreme biochemical properties, low immunogenicity, and the ability to bind targets with high affinity and selectivity. While many CDPs have potential and confirmed therapeutic uses, synthesis of CDPs is a challenge. Recent advances have made the recombinant expression of CDPs a viable alternative to chemical synthesis. Moreover, identifying CDPs that can be expressed in mammalian cells is crucial in predicting their compatibility with gene therapy and mRNA therapy. Currently, we lack the ability to identify CDPs that will express recombinantly in mammalian cells without labour intensive experimentation. To address this, we developed CysPresso, a novel machine learning model that predicts recombinant expression of CDPs based on primary sequence. Results: We tested various protein representations generated by deep learning algorithms (SeqVec, proteInfer, AlphaFold2) for their suitability in predicting CDP expression and found that AlphaFold2 representations possessed the best predictive features. We then optimized the model by concatenation of AlphaFold2 representations, time series transformation with random convolutional kernels, and dataset partitioning. Conclusion: Our novel model, CysPresso, is the first to successfully predict recombinant CDP expression in mammalian cells and is particularly well suited for predicting recombinant expression of knottin peptides. When preprocessing the deep learning protein representation for supervised machine learning, we found that random convolutional kernel transformation preserves more pertinent information relevant for predicting expressibility than embedding averaging. Our study showcases the applicability of deep learning-based protein representations, such as those provided by AlphaFold2, in tasks beyond structure prediction.

bioinformatics↗

bioRxiv2022

A high-speed, modular display system for diverse neuroscience applications

Visual stimulation of animals in the laboratory is a powerful technique for studying sensory control of complex behaviors. Since commercial displays are optimized for human vision, we established a novel display system based on custom-built modular LED panels that provides millisecond refresh, precise synchronization, customizable color combinations, and varied display configurations. This system simplifies challenging experiments. With variants of this display, we probed the speed limits of motion vision and examined the role of color vision in behavioral experiments with tethered flying Drosophila. Using 2-photon calcium imaging, we comprehensively mapped the tuning of visual projection neurons across the flys field of view. Finally, using real-time behavior analysis, we developed low-latency interactive virtual environments and found that flying flies can independently control their navigation along two dimensions. This display system uniquely addresses most technical challenges of small animal vision experiments and is thoroughly documented for replicability.

neuroscience↗

bioRxiv2022

Ribosome stalling during selenoprotein translation exposes a ferroptosis vulnerability in cancer

Ferroptosis is a regulated, iron-dependent form of necrosis that is triggered by the accumulation of oxidatively damaged phospholipids1-3. Glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting phospholipid hydroperoxides into non-toxic lipid alcohols4, 5. Ferroptosis has been implicated in the pathology of several degenerative conditions and inhibiting GPX4 activity has emerged as a therapeutic strategy to induce cancer cell death1, 2. However, many cancer cell lines are resistant to GPX4 inhibition6, and the mechanisms that regulate GPX4 activity and ferroptosis resistance remain incompletely understood. Here, employing a synthetic lethal CRISPR-Cas9 screen in a triple negative breast cancer (TNBC) cell line, we identify LRP8 (also known as ApoER2) as a ferroptosis resistance factor. LRP8 is upregulated in cancer, and we find that it promotes ferroptosis resistance in cancer cells in both 2-dimensional (2-D) cell culture and 3-dimensional (3-D) spheroid models. Mechanistically, loss of LRP8 decreases cellular selenium levels, resulting in the reduced expression of a subset of selenoproteins, including GPX4. Remarkably, the reduction in GPX4 is not due to the classic hierarchical selenoprotein regulatory program7, 8. Instead, our findings demonstrate that the translation of GPX4 is severely impaired in the selenium-deficient LRP8 knockout (KO) cells due to extensive ribosome stalling at the inefficiently decoded GPX4 selenocysteine (SEC) UGA codon, which results in ribosome collisions and early translation termination. Thus, our findings reveal ribosome stalling and collisions during GPX4 translation as targetable ferroptosis vulnerabilities in cancer cells.

cancer biology↗

bioRxiv2022

Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR

Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA secondary structure and nucleotide modifications can both interfere with cDNA synthesis by commonly-used reverse transcriptases (RTs). Here, we benchmark a recently-developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We find that OTTR robustly captures full-length tRNAs in budding yeast and in mouse testis, with relatively low levels of premature termination at known barriers - 1-methylguanine, N2,N2-dimethylguanine, and 1-methyladenine - to typical reverse transcriptases. Moreover, these and several other nucleotide modifications leave misincorporation signatures in OTTR datasets which enables their detection without any additional protocol steps. Turning to analysis of small RNAs such as tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to "ground truth" Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRFs - including both 5 and 3 tRNA halves derived from the majority of tRNAs - than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.

genomics↗

bioRxiv2021

Robust T cell activation requires an eIF3-driven burst in T cell receptor translation

Activation of T cells requires a rapid surge in cellular protein synthesis. However, the role of translation initiation in the early induction of specific genes remains unclear. Here we show human translation initiation factor eIF3 interacts with select immune system related mRNAs including those encoding the T cell receptor (TCR) subunits TCRA and TCRB. Binding of eIF3 to the TCRA and TCRB mRNA 3-untranslated regions (3-UTRs) depends on CD28 coreceptor signaling and regulates a burst in TCR translation required for robust T cell activation. Use of the TCRA or TCRB 3-UTRs to control expression of an anti-CD19 chimeric antigen receptor (CAR) improves the ability of CAR-T cells to kill tumor cells in vitro. These results identify a new mechanism of eIF3-mediated translation control that can aid T cell engineering for immunotherapy applications.

molecular biology↗

bioRxiv2020

CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes

To realize the promise of CRISPR/Cas9-based genetics, approaches are needed to quantify a specific, molecular phenotype across genome-wide libraries of genetic perturbations. We address this challenge by profiling transcriptional, translational, and post-translational reporters using CRISPR interference with barcoded expression reporter sequencing (CiBER-seq). Our barcoding approach connects an entire library of guides to their individual phenotypic consequences using pooled sequencing. We show that CiBER-seq profiling fully recapitulates the integrated stress response (ISR) pathway in yeast. Genetic perturbations causing uncharged tRNA accumulation activated ISR reporter transcription. Surprisingly, tRNA insufficiency also activated the reporter, independent of the Gcn2 kinase that senses uncharged tRNAs. By uncovering alternate triggers for ISR activation, we illustrate how precise, comprehensive CiBER-seq profiling provides a powerful and broadly applicable tool for dissecting genetic networks.

genomics↗

bioRxiv2020

Amygdala-cortical control of striatal plasticity drives the acquisition of goal-directed action.

The acquisition of goal-directed action requires the encoding of specific action-outcome associations involving plasticity in the posterior dorsomedial striatum (pDMS). We first investigated the relative involvement of the major inputs to the pDMS argued to be involved in this learning-related plasticity, from prelimbic prefrontal cortex (PL) and from the basolateral amygdala (BLA). Using ex vivo optogenetic stimulation of PL or BLA terminals in pDMS, we found that goal-directed learning potentiated the PL input to direct pathway spiny projection neurons (dSPNs) bilaterally but not to indirect pathway neurons (iSPNs). In contrast, learning-related plasticity was not observed in the direct BLA-pDMS pathway. Using toxicogenetics, we ablated BLA projections to either pDMS or PL and found that only the latter was necessary for goal-directed learning. Importantly, transient inactivation of the BLA during goal-directed learning prevented the PL-pDMS potentiation of dSPNs, establishing that the BLA input to the PL is necessary for the corticostriatal plasticity underlying goal-directed learning.

neuroscience↗

bioRxiv2019

Dynamics of T cell activation mediated by eIF3 interactions with T cell receptor mRNAs

Activation of T cells requires a global surge in cellular protein synthesis, accompanied by a large increase in translation initiation1-4. A central component of the translation initiation machinery-the multi-subunit eukaryotic initiation factor 3 (eIF3)-is rapidly turned on when quiescent T cells are stimulated3. However, the precise role eIF3 plays in activated T cells is not known. Using a global transcriptome crosslinking approach, we show human eIF3 interacts with a distinct set of mRNAs in activated Jurkat cells. A subset of these mRNAs, including those encoding the T cell receptor (TCR) subunits TCRA and TCRB, crosslink to eIF3 across the entire length of the mRNA. The TCRA and TCRB mRNAs do not co-localize with translationally repressed environments of P-bodies or stress granules but form distinct granules, potentially acting as translation "hot-spots." T cell activation through CD28 causes a burst of TCR translation controlled by elements in the 3-untranslated regions (3-UTRs) of the TCRA and TCRB mRNAs that directly contact eIF3 and that are required for T cell activity. These results highlight a new role for eIF3 in regulating the translation dynamics of the TCR and provide insights that can guide the engineering of T cells used in cell immunotherapy applications.

molecular biology↗

bioRxiv2019

Isoform specificity of PKMs during long-term facilitation in Aplysia is mediated through stabilization by KIBRA

Persistent activity of protein kinase M (PKM), the truncated form of protein kinase C (PKC), can maintain long-term changes in synaptic strength in many systems including the hermaphrodite marine mollusk, Aplysia californica. Moreover, different types of long-term facilitation (LTF) in cultured Aplysia sensorimotor synapses rely on the activities of different PKM isoforms in the presynaptic sensory neuron and postsynaptic motor neuron. When the atypical PKM isoform was required, the kidney and brain expressed adaptor protein (KIBRA) was also required. Here, we explore how this isoform specificity is established. We find that PKM overexpression in the motor neuron, but not the sensory neuron, is sufficient to increase synaptic strength and that this activity is not isoform specific. KIBRA is not the rate-limiting step in facilitation since overexpression of KIBRA is neither sufficient to increase synaptic strength, nor to prolong a form of PKM-dependent intermediate synaptic facilitation. However, the isoform specificity of dominant negative (DN)-PKMs to erase LTF is correlated with isoform specific competition for stabilization by KIBRA. We identify a new conserved region of KIBRA. Different splice isoforms in this region stabilize different PKMs based on the isoform-specific sequence of an alpha-helix handle in the PKMs. Thus, specific stabilization of distinct PKMs by different isoforms of KIBRA can explain the isoform specificity of PKMs during LTF in Aplysia.\n\nSignificance StatementLong lasting changes in synaptic plasticity associated with memory formation are maintained by persistent protein kinases. We have previously shown in the Aplysia sensorimotor model that distinct isoforms of persistently active protein kinase Cs (PKMs) maintain distinct forms of long-lasting synaptic changes, even when both forms are expressed in the same motor neuron. Here, we show that, while the effects of overexpression of PKMs in not isoform specific, isoform specificity is defined by a handle helix in PKMs that confers stabilization by distinct splice forms in a previously undefined domain of the adaptor protein KIBRA. Thus, we define new regions in both KIBRA and PKMs that define the isoform specificity for maintaining synaptic strength in distinct facilitation paradigms.

neuroscience↗