Biology subjects

Duernberger, G.

Publications and source records attributed to Duernberger, G..

5 recordsLinked to original sources

bioRxiv2023

eIF4E1b is a non-canonical eIF4E required for maternal mRNA dormancy

Maternal mRNAs are essential for protein synthesis during oogenesis and early embryogenesis. To adapt translation to specific needs during development, maternal mRNAs are translationally repressed by shortening the polyA tails. While mRNA deadenylation is associated with decapping and degradation in somatic cells, maternal mRNAs with short polyA tails are stable. Here we report an essential role for the germline-specific paralog of the mRNA cap-binding factor eIF4E, known as eIF4E1b, in the storage and repression of maternal mRNAs with short polyA tails. eIF4E1b binds to the mRNA cap and is targeted to ribonucleoprotein complexes through its direct interaction with eIF4ENIF1/4E-T. In early embryos, eIF4E1b binds to a specific set of translationally repressed mRNAs with short or no polyA tails, such as histone mRNAs, which are translated later on during embryogenesis. Consistent with an important role in maternal mRNA dormancy, mutation of eIF4E1b in zebrafish impairs female germline development. Understanding the mechanism and function of eIF4E1B provides new insights into fundamental post-transcriptional regulatory principles governing early vertebrate development.

developmental biology↗

bioRxiv2022

Integrated transcriptome and proteome analysis in human brain organoids reveals translational regulation of ribosomal proteins.

During development of the human cerebral cortex, multipotent neural progenitors generate excitatory neurons and glial cells. Investigations of the transcriptome and epigenome have revealed important gene regulatory networks underlying this crucial developmental event. However, the post-transcriptional control of gene expression and protein abundance during human corticogenesis remains poorly understood. We addressed this issue by using human telencephalic brain organoids grown using a dual reporter cell line to isolate neural progenitors and neurons and performed cell class and developmental stage-specific transcriptome and proteome analysis. Integrating the two datasets revealed modules of gene expression during human corticogenesis. Investigation of one such module uncovered mTOR-mediated regulation of translation of the 5TOP element-enriched translation machinery in early progenitor cells. We show that in early progenitors partial inhibition of the translation of ribosomal genes prevents precocious translation of differentiation markers. Overall, our multiomics approach reveals novel posttranscriptional regulatory mechanisms crucial for the fidelity of cortical development.

developmental biology↗

bioRxiv2022

Robust and easy-to-use one pot workflow for label free single cell proteomics

The analysis of ultra-low input samples or even individual cells is essential to answering a multitude of biomedical questions, but current proteomic workflows are limited in their sensitivity and reproducibility. Here we report a comprehensive workflow that includes optimized strategies for all steps from cell lysis to data analysis. Thanks to convenient to handle 1 L sample volume and standardized 384 well plates the workflow is easy for even novice users to implement. At the same time, it can be performed semi-automatized using the CellenONE(R), which allows for highest reproducibility. To achieve high throughput, ultrashort gradient lengths down to 5 min were tested using advanced -pillar columns. Data-dependent acquisition (DDA), wide-window acquisition (WWA) and data-independent acquisition (DIA), and commonly used advanced data-analysis algorithms were benchmarked. Using DDA, 1790 proteins covering a dynamic range of four orders of magnitude were identified in a single cell. Using DIA, proteome coverage increased to more than 2200 proteins identified from single cell level input in a 20-min active gradient. The workflow enabled differentiation of two cell lines, demonstrating its suitability to cellular heterogeneity determination.

biochemistry↗

bioRxiv2021

Analysis of 8000 proteins and reduced carry over significantly increase the throughput of single-shot proteomics

In the field of LC-MS based proteomics, increases in sampling depth and proteome coverage have mainly been accomplished by rapid advances in mass spectrometer technology. The comprehensiveness and quality of data that can be generated do however also depend on the performance provided by nano liquid chromatography (nanoLC) separations. Proper selection of reversed-phase separation columns can be important to provide the MS instrument with peptides at the highest possible concentration and separated at the highest possible resolution. In the current contribution, we evaluate the use of prototype generation 2 PAC nanoLC columns which use C18 functionalized superficially porous micro pillars as a stationary phase. When comparing to traditionally used fully porous silica stationary phases, more precursors could be characterized when performing single shot data-dependent LC-MS/MS analyses of a human cell line tryptic digest. Up to 30% more protein groups and 60% more unique peptides were identified for short gradients (10 min) and limited sample amounts (10-100 ng of cell lysate digest). With LC-MS gradient times of 10, 60, 120 and 180 min, we respectively identified 2252, 6513, 7382 and 8174 protein groups with 25, 500, 1000 and 2000 ng of sample loaded on column. Reduction of sample carry over to the next run (up to 2 to 3%) and decreased levels of methionine oxidation (up to 3-fold) were identified as additional figures of merit. When analyzing a disuccinimidyl dibutyric urea (DSBU) crosslinked synthetic library, 29 to 59 more unique crosslinked peptides could be identified at a experimentally validated false discovery rate (FDR) of 1-2%.

biochemistry↗

bioRxiv2019

ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesinSTAG1 from WAPL

Eukaryotic genomes are folded into loops. It is thought that these are formed by cohesin complexes via extrusion, either until loop expansion is arrested by CTCF or until cohesin is removed from DNA by WAPL. Although WAPL limits cohesins chromatin residence time to minutes, it has been reported that some loops exist for hours. How these loops can persist is unknown. We show that during G1-phase, mammalian cells contain acetylated cohesinSTAG1 which binds chromatin for hours, whereas cohesinSTAG2 binds chromatin for minutes. Our results indicate that CTCF and the acetyltransferase ESCO1 protect a subset of cohesinSTAG1 complexes from WAPL, thereby enable formation of long and presumably long-lived loops, and that ESCO1, like CTCF, contributes to boundary formation in chromatin looping. Our data are consistent with a model of nested loop extrusion, in which acetylated cohesinSTAG1 forms stable loops between CTCF sites, demarcating the boundaries of more transient cohesinSTAG2 extrusion activity.

cell biology↗