Biology subjects

Marr, M. T.

Publications and source records attributed to Marr, M. T..

6 recordsLinked to original sources

bioRxiv2026

Acute EIF4G1 depletion reveals transcript-wide determinants of initiation factor dependence and activates initiation-specific ribosome quality control

Canonical cap-dependent translation initiation requires the eIF4F complex, in which the scaffold protein EIF4G1 bridges cap-bound EIF4E, the RNA helicase EIF4A, and ribosome-associated EIF3. Using an inducible degron to acutely deplete EIF4G1 in human cells, we combined polysome profiling with mRNA-seq to quantify transcript-specific relative polysome association (RPA). EIF4G1 depletion causes heavy polysome collapse with reciprocal 80S monosome accumulation and activates initiation-specific ribosome quality control (iRQC), evidenced by RPS3 mono-ubiquitination enriched on 80S monosomes. Using RPA analysis we find the ribosome association of most transcripts is unaffected. However, we identify 1,250 mRNAs that are sensitive to EIF4G1 loss and 1,663 mRNAs that are translationally enhanced under the same conditions. As a class, the enhanced transcripts tend to be longer and have a higher GC content in the coding sequence relative to sensitive or unaffected transcripts. A synthetic reporter confirms that elevated CDS GC content can promote translation upon EIF4G1 depletion. These results suggest that the architecture of the entire transcript, beyond the 5'UTR, affects EIF4G1 usage and that removing EIF4G1 triggers iRQC consistent with an altered initiation architecture.

molecular biology↗

bioRxiv2024

Structure-informed mutagenesis identifies a conserved region critical for mouse insulin receptor 5'UTR IRES function

Cells under stress shift their proteome by repressing cap-dependent translation initiation. RNA elements called internal ribosome entry sites (IRES) can allow key cellular transcripts to remain efficiently translated to support an effective stress response. Well- characterized IRESes depend on RNA structures that reduce the protein requirements for translation initiation, thus circumventing translation inhibition. We have previously determined that the insulin receptor 5 untranslated region (5UTR) possesses a capacity for IRES activity that is conserved from insects to mammals. There are several prominent examples of viral IRES structures solved in solution; however, the RNA secondary structures of cellular IRESes remain mostly elusive, especially in vivo. Here we probe the secondary structure of the Insr 5UTR IRES in tandem with two well-studied viral IRESes from Hepatitis C virus (HCV) and Encephalomyocarditis virus (EMCV) using dimethyl sulfate mutational profiling by sequencing (DMS-MaPseq) in cells and in vitro. We find that the viral IRES structures in cells are consistent with their known in vitro structures and that significant linearization of these well-studied IRESes occurs in the region surrounding their translation start codon in cells. Using the concurrent DMS- MaPseq probing as a constraint, we present a model of the mouse insulin receptor (Insr) 5UTR. With this model as a guide, we employed a mutation strategy which allowed us to identify a conserved segment distal from the translation start codon as critical for Insr IRES function. This knowledge informed the design of a minimal IRES element with equivalent activity to the full-length Insr 5UTR across translation contexts. BackgroundThe RNA structural requirements for cap-independent translation initiation facilitated by the Mus musculus insulin receptor (Insr) 5 untranslated region (5UTR) are unknown. ResultsRNA secondary structure probing of the Insr 5UTR in cells provides a folding model used to identify elements required for cap-independent translation initiation. ConclusionA small region of the Insr 5UTR distal to the translation start codon is necessary for cap-independent translation initiation and is fully sufficient in the proper structural context.

molecular biology↗

bioRxiv2022

The Insulin receptor and Insulin like growth factor receptor 5 UTRs support translation initiation independently of eIF4G1

Protein synthesis is tightly regulated under stress conditions where energy may be scarce. Despite global repression of translation, some transcripts remain actively translated in order for the cell to be able to respond to the insult or prepare to quickly return normal cellular function after the stress ends. For the insulin receptor (Insr) and insulin-like growth factor receptor (Igf1r) transcripts this translation is mediated by an internal ribosome entry site (IRES) in their 5UTRs that functions independently of eukaryotic initiation factor 4A (eIF4A) and eIF4E. Here we show that these cellular IRESes are also able to promote translation independently of the scaffolding protein eIF4G1 both in vitro and in the cell. BackgroundIRES mediated translation initiation requires a different repertoire of factors than canonical cap-dependent translation. ResultsTreatments that inhibit the canonical translation factor eIF4G1 have little or no effect on the ability of the Insr and Igf1r cellular IRESes to promote translation. ConclusionTranscripts for two cellular receptors contain RNA elements that facilitate translation initiation without intact eIF4G1 SignificanceCellular IRES mechanisms may resemble viral type III IRESes allowing them to promote translate with a limited number of initiation factors allowing them to work under stress conditions when canonical translation is repressed.

molecular biology↗

bioRxiv2022

Artificially stimulating retrotransposon activity increases mortality and accelerates a subset of aging phenotypes in Drosophila

Transposable elements (TE) are mobile sequences of DNA that can become transcriptionally active as an animal ages. Whether TE activity is simply a byproduct of heterochromatin breakdown or can contribute towards the aging process is not known. Here we place the TE gypsy under the control of the UAS GAL4 system to model TE activation during aging. We find that increased TE activity shortens the lifespan of male D. melanogaster. The effect is only apparent in middle aged animals. The increase in mortality is not seen in young animals. An intact reverse transcriptase is necessary for the decrease in lifespan implicating a DNA mediated process in the effect. The decline in lifespan in the active gypsy flies is accompanied by the acceleration of a subset of aging phenotypes. TE activity increases sensitivity to oxidative stress and promotes a decline in circadian rhythmicity. The overexpression of the Forkhead-box O family (FOXO) stress response transcription factor can partially rescue the detrimental effects of increased TE activity on lifespan. Our results provide evidence that active TEs can behave as effectors in the aging process and suggest a potential novel role for dFOXO in its promotion of longevity in D. melanogaster.

molecular biology↗

bioRxiv2017

The Ras-like GTPase Rem2 is a potent endogenous inhibitor of calcium/calmodulin-dependent kinase II activity

CaMKII is a well-characterized, abundant protein kinase that regulates a diverse set of functions in a tissue specific manner. For example, in heart muscle, CaMKII regulates Ca2+ homeostasis while in neurons CaMKII regulates activity-dependent dendritic remodeling and Long Term Potentiation (LTP), a biological correlate of learning and memory. Previously, we identified the noncanonical GTPase Rem2 as a critical regulator of dendrite branching and synapse formation in the vertebrate nervous system. Here, we report that Rem2 directly interacts with CaMKII and potently inhibits the activity of the intact holoenzyme, a previously undescribed function for the Rem2 protein. To date, only one other endogenous inhibitor of CaMKII has been described: CaMKIIN, which blocks CaMKII activity through binding to the catalytic domain. Our data suggest that Rem2 inhibits CaMKII through a novel mechanism, as inhibition requires the presence of the association domain of CaMKII. Our biochemical finding that Rem2 is a direct, endogenous inhibitor of CaMKII activity, coupled with known functions of Rem2 in neurons, provides a framework which will enable future experiments probing the physiological role of CaMKII inhibition in a cellular context.

biochemistry↗

bioRxiv2017

The integrated stress response pathway activates 4E-BP to bias mRNA translation and boost antimicrobial peptide synthesis in response to bacterial infection

Pathogenic bacterial infection imposes considerable cellular stress on the host and often leads to attenuation of mRNA translation. In this translation-suppressive environment, it is unclear how the host synthesizes various antimicrobial peptides (AMPs) to mount innate immune response. Here, we use Drosophila as a model to demonstrate that AMP production during infection relies on a translation bias mechanism mediated by the inhibitor of cap-dependent translation 4E-BP (Drosophila Thor), and the AMP 5UTRs that can undergo cap-independent translation. We found that 4E-BP is induced upon infection with the pathogenic bacteria Ecc15 by the stress-responsive transcription factor ATF4, and its upstream kinase GCN2. Moreover, loss of gcn2, atf4 or 4e-bp compromised immunity against Ecc15. In 4E-BP mutants, the transcriptional induction of AMPs after infection was unaffected, while the protein levels of AMPs were substantially reduced in their hemolymph. Analysis of the 5UTRs of AMPs using cell-based bicistronic reporters and in vitro translation analysis indicated that AMPs are translated in a cap-independent mechanism. Analysis of bicistronic reporters in the presence of 4E-BP indicate that infection enhances cap-independent translational activity associated with AMP 5UTRs, accounting for enhanced AMP translation during infection.\n\nHighlights O_LI4E-BP is transcriptionally induced by GCN2/ATF signaling in response to bacterial infection\nC_LIO_LI4E-BP mutants show unaltered antimicrobial peptide (AMP) transcript levels, but have reduced AMP translation\nC_LIO_LIAMP 5UTRs are translated cap-independently\nC_LIO_LITranslation bias by 4E-BP drives cap-independent AMP translation\nC_LI

immunology↗