Genome-wide conditional degron libraries for functional genomics.

Functional genomics with libraries of knockout alleles is limited to non-essential genes and convoluted by the potential accumulation of suppressor mutations in knockout backgrounds, which can lead to erroneous functional annotations. To address these limitations, we constructed genome-wide libraries of conditional alleles based on the auxin-inducible degron (AID) system for inducible degradation of AID-tagged proteins in the budding yeast Saccharomyces cerevisiae. First, we determined that N-terminal tagging is at least twice as likely to inadvertently impair protein function across the proteome.

Building yeast libraries to dissect terminal degrons with fluorescent timers.

Selective degradation of unnecessary or abnormal proteins by the ubiquitin-proteasome system is an essential part of proteostasis. Ubiquitin ligases recognize substrates of selective protein degradation and modify them with polyubiquitin chains, which mark them for proteasomal degradation. Substrate recognition by ubiquitin ligases often involves degradation signals or degrons, which are typically short linear motifs found in intrinsically disordered regions, e.

Genetic requirements for repair of lesions caused by single genomic ribonucleotides in S phase.

Single ribonucleoside monophosphates (rNMPs) are transiently present in eukaryotic genomes. The RNase H2-dependent ribonucleotide excision repair (RER) pathway ensures error-free rNMP removal. In some pathological conditions, rNMP removal is impaired.

SWR1 chromatin remodeling complex prevents mitotic slippage during spindle position checkpoint arrest.

Faithful chromosome segregation in budding yeast requires correct positioning of the mitotic spindle along the mother to daughter cell polarity axis. When the anaphase spindle is not correctly positioned, a surveillance mechanism, named as the spindle position checkpoint (SPOC), prevents the progression out of mitosis until correct spindle positioning is achieved. How SPOC works on a molecular level is not well understood.

Up-regulation of ubiquitin-proteasome activity upon loss of NatA-dependent N-terminal acetylation.

N-terminal acetylation is a prominent protein modification, and inactivation of N-terminal acetyltransferases (NATs) cause protein homeostasis stress. Using multiplexed protein stability profiling with linear ubiquitin fusions as reporters for the activity of the ubiquitin proteasome system, we observed increased ubiquitin proteasome system activity in NatA, but not NatB or NatC mutants. We find several mechanisms contributing to this behavior.

Quality control of mislocalized and orphan proteins.

A healthy and functional proteome is essential to cell physiology. However, this is constantly being challenged as most steps of protein metabolism are error-prone and changes in the physico-chemical environment can affect protein structure and function, thereby disrupting proteome homeostasis. Among a variety of potential mistakes, proteins can be targeted to incorrect compartments or subunits of protein complexes may fail to assemble properly with their partners, resulting in the formation of mislocalized and orphan proteins, respectively.

Exploring whole-genome duplicate gene retention with complex genetic interaction analysis.

Whole-genome duplication has played a central role in the genome evolution of many organisms, including the human genome. Most duplicated genes are eliminated, and factors that influence the retention of persisting duplicates remain poorly understood. We describe a systematic complex genetic interaction analysis with yeast paralogs derived from the whole-genome duplication event.

(Photo)convert to pooled visual screening.

Pooled genetic screening is a powerful method to systematically link genotype to phenotype and gain insights into biological processes, but applying it to visual phenotypes such as cell morphology or protein localization has remained a challenge. In their recent work, Fowler and colleagues (Hasle et al, 2020) describe an elegant approach for high-throughput cell sorting according to visual phenotypes based on selective photoconversion. This allows combining the advantages of high-content phenotyping by fluorescence microscopy with the efficiency of pooled screening to dissect complex phenotypes.

Ubc13-Mms2 cooperates with a family of RING E3 proteins in budding yeast membrane protein sorting.

Polyubiquitin chains linked via lysine (K) 63 play an important role in endocytosis and membrane trafficking. Their primary source is the ubiquitin protein ligase (E3) Rsp5/NEDD4, which acts as a key regulator of membrane protein sorting. The heterodimeric ubiquitin-conjugating enzyme (E2), Ubc13-Mms2, catalyses K63-specific polyubiquitylation in genome maintenance and inflammatory signalling.

Increased Mortality and Vascular Phenotype in a Knock-In Mouse Model of Retinal Vasculopathy With Cerebral Leukoencephalopathy and Systemic Manifestations.

Background and Purpose- Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is an autosomal dominant small vessel disease caused by C-terminal frameshift mutations in the gene that encodes the major mammalian 3' to 5' DNA exonuclease. RVCL-S is characterized by vasculopathy, especially in densely vascularized organs, progressive retinopathy, cerebral microvascular disease, white matter lesions, and migraine, but the underlying mechanisms are unknown. Methods- Homozygous transgenic RVCL-S knock-in mice expressing a truncated Trex1 (three prime repair exonuclease 1) protein (similar to what is seen in patients) and wild-type littermates, of various age groups, were subjected to (1) a survival analysis, (2) in vivo postocclusive reactive hyperemia and ex vivo Mulvany myograph studies to characterize the microvascular and macrovascular reactivity, and (3) experimental stroke after transient middle cerebral artery occlusion with neurological deficit assessment.

Evaluation of acute esophageal radiation-induced damage using magnetic resonance imaging: a feasibility study in mice.

Background: Thoracic and head and neck cancer radiation therapy (RT) can cause damage to nearby healthy organs such as the esophagus, causing acute radiation-induced esophageal damage (ARIED). A non-invasive method to detect and monitor ARIED can facilitate optimizing RT to avoid ARIED while improving local tumor control. Current clinical guidelines are limited to scoring the esophageal damage based on the symptoms of patients.

Cooperation of mitochondrial and ER factors in quality control of tail-anchored proteins.

Tail-anchored (TA) proteins insert post-translationally into the endoplasmic reticulum (ER), the outer mitochondrial membrane (OMM) and peroxisomes. Whereas the GET pathway controls ER-targeting, no dedicated factors are known for OMM insertion, posing the question of how accuracy is achieved. The mitochondrial AAA-ATPase Msp1 removes mislocalized TA proteins from the OMM, but it is unclear, how Msp1 clients are targeted for degradation.

Micro cone beam computed tomography for sensitive assessment of radiation-induced late lung toxicity in preclinical models.

Background And Purpose: Preclinical models are much needed to assess the effect of novel radio-sensitizers or mitigators on radiation dose limiting lung toxicity. Albeit showing radiation-induced lung pathologies, current mouse models lack the sensitivity to do so. Using micro image-guided radiotherapy (µIGRT) techniques, we aimed to establish murine models which enable the sensitive detection of lung damage aggravation and characterized functional, radiological and histological responses.

Optical coherence tomography to detect acute esophageal radiation-induced damage in mice: A validation study.

Radiation therapy for patients with non-small-cell lung cancer is hampered by acute radiation-induced toxicity in the esophagus. This study aims to validate that optical coherence tomography (OCT), a minimally invasive imaging technique with high resolution (~10 μm), is able to visualize and monitor acute radiation-induced esophageal damage (ARIED) in mice. We compare our findings with histopathology as the gold standard.

Genome-wide C-SWAT library for high-throughput yeast genome tagging.

Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae open reading frames (ORFs). In 5,661 strains, we inserted an acceptor module after each ORF that can be efficiently replaced with tags or regulatory elements. We validated the library with targeted sequencing and tagged the proteome with bright fluorescent proteins to quantify the effect of heterologous transcription terminators on protein expression and to localize previously undetected proteins.

Determinants of the cytosolic turnover of mitochondrial intermembrane space proteins.

Background: The proteome of mitochondria comprises mostly proteins that originate as precursors in the cytosol. Before import into the organelle, such proteins are exposed to cytosolic quality control mechanisms. Multiple lines of evidence indicate a significant contribution of the major cytosolic protein degradation machinery, the ubiquitin-proteasome system, to the quality control of mitochondrial proteins.

Mapping Degradation Signals and Pathways in a Eukaryotic N-terminome.

Most eukaryotic proteins are N-terminally acetylated. This modification can be recognized as a signal for selective protein degradation (degron) by the N-end rule pathways. However, the prevalence and specificity of such degrons in the proteome are unclear.

Transit time mapping in the mouse brain using time-encoded pCASL.

The cerebral blood flow (CBF) is a potential biomarker for neurological disease. However, the arterial transit time (ATT) of the labeled blood is known to potentially affect CBF quantification. Furthermore, ATT could be an interesting biomarker in itself, as it may reflect underlying macro- and microvascular pathologies.