Expression and subcellular localization of USH1C/harmonin in human retina provides insights into pathomechanisms and therapy.

Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones and outer segments (OS) of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in PRCs and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology and development of gene therapy treatment(s).

Cell cycle progression and transmitotic apoptosis resistance promote escape from extrinsic apoptosis.

Extrinsic apoptosis relies on TNF-family receptor activation by immune cells or receptor-activating drugs. Here, we monitored cell cycle progression at a resolution of minutes to relate apoptosis kinetics and cell-to-cell heterogeneities in death decisions to cell cycle phases. Interestingly, we found that cells in S phase delay TRAIL receptor-induced death in favour of mitosis, thereby passing on an apoptosis-primed state to their offspring. This translates into two distinct fates, apoptosis execution post mitosis or cell survival from inefficient apoptosis. Transmitotic resistance is linked to Mcl-1 upregulation and its increased accumulation at mitochondria from mid-S phase onwards, which allows cells to pass through mitosis with activated caspase-8, and with cells escaping apoptosis after mitosis sustaining sublethal DNA damage. Antagonizing Mcl-1 suppresses cell cycle-dependent delays in apoptosis, prevents apoptosis-resistant progression through mitosis and averts unwanted survival after apoptosis induction. Cell cycle progression therefore modulates signal transduction during extrinsic apoptosis, with Mcl-1 governing decision making between death, proliferation and survival. Cell cycle progression thus is a crucial process from which cell-to-cell heterogeneities in fates and treatment outcomes emerge in isogenic cell populations during extrinsic apoptosis. This article has an associated First Person interview with the first author of the paper.

Pathogenic Variants in USH1G/SANS Alter Protein Interaction with Pre-RNA Processing Factors PRPF6 and PRPF31 of the Spliceosome.

Pre-mRNA splicing is an essential process orchestrated by the spliceosome, a dynamic complex assembled stepwise on pre-mRNA. We have previously identified that USH1G protein SANS regulates pre-mRNA splicing by mediating the intranuclear transfer of the spliceosomal U4/U6.U5 tri-snRNP complex. During this process, SANS interacts with the U4/U6 and U5 snRNP-specific proteins PRPF31 and PRPF6 and regulates splicing, which is disturbed by variants of USH1G/SANS causative for human Usher syndrome (USH), the most common form of hereditary deaf-blindness. Here, we aim to gain further insights into the molecular interaction of the splicing molecules PRPF31 and PRPF6 to the CENTn domain of SANS using fluorescence resonance energy transfer assays in cells and in silico deep learning-based protein structure predictions. This demonstrates that SANS directly binds via two distinct conserved regions of its CENTn to the two PRPFs. In addition, we provide evidence that these interactions occur sequentially and a conformational change of an intrinsically disordered region to a short α-helix of SANS CENTn2 is triggered by the binding of PRPF6. Furthermore, we find that pathogenic variants of USH1G/SANS perturb the binding of SANS to both PRPFs, implying a significance for the USH1G pathophysiology.

An overview of the bacterial SsrA system modulating intracellular protein levels and activities.

In bacteria, the truncated forms of mRNAs, which usually lack a stop codon, are occasionally generated by premature termination of gene transcription and/or endo- or exonucleolytic cleavage events. Ribosomes proceeding on these molecules stall at the 3' end of the chain and are rescued by a widely distributed mechanism known as trans-translation, which includes two essential elements, ssrA RNA (a special RNA) and SmpB (a small protein). Through this mechanism, the polypeptides translated from truncated mRNAs are marked by a short peptide, known as SsrA tag, at their C-termini and directed to the specific endogenous proteases for C-terminal proteolysis. Based on the deep understanding of the SsrA tagging and degradation mechanisms, recently a series of SsrA-based genetic tools have been developed for gene regulation on the level of post-translation. They are successfully applied for controllable regulation of biological circuits in bacteria. In the present article, we systematically summarize the history, structural characteristics, and functional mechanisms of the SsrA tagging and degrading machineries, as well as their technical uses and limitations.Key Points• SsrA system plays an important role in ribosome rescue in bacteria.• SsrA-based genetic tools are useful for controlling protein levels and activities.

Engineering a Controllable Targeted Protein Degradation System and a Derived OR-GATE-Type Inducible Gene Expression System in Synechococcus elongatus PCC 7942.

Cyanobacteria are important model organisms for exploring the mechanisms of photosynthesis and are considered as promising microbial platforms for photosynthetic biomanufacturing. The development of efficient cyanobacteria cell factories requires efficient and convenient tools to dynamically regulate and manipulate target proteins, modules, and pathways. Targeted protein degradation is important to achieve rapid responses of cellular metabolic networks to artificial or environmental signals, and there are currently limited approaches to induce protein degradation in cyanobacteria. In this work, we developed an Escherichia coli sourced ssrA-tagging system in an important cyanobacteria strain, Synechococcus elongatus PCC 7942, to achieve inducible degradation of target proteins. A modified version of the E. coli ssrA tag (ssrADAS) proved to be immune to the native ClpXP system in Synechococcus elongatus PCC 7942, while induced expression of the E. coli sourced adaptor SspB and ClpXP resulted in effective degradation of the tagged proteins. Compared to the previously developed down-regulation approaches, the inducible ssrADAS-SspB-ClpXPEc system facilitated the smart and rapid degradation of target proteins in PCC7942 cells at different growth stages. Furthermore, when used to regulate the degradation of LacI, the repressor element of LacO-LacI transcription regulation system, an efficient and stringent inducible gene expression system was obtained based on an OR-GATE type genetic circuit design. The tools developed in this work expanded the cyanobacteria synthetic biology toolbox and will facilitate the success of future dynamic metabolic engineering.

Phosphorylations of the Abutilon Mosaic Virus Movement Protein Affect Its Self-Interaction, Symptom Development, Viral DNA Accumulation, and Host Range.

The genome of bipartite geminiviruses in the genus Begomovirus comprises two circular DNAs: DNA-A and DNA-B. The DNA-B component encodes a nuclear shuttle protein (NSP) and a movement protein (MP), which cooperate for systemic spread of infectious nucleic acids within host plants and affect pathogenicity. MP mediates multiple functions during intra- and intercellular trafficking, such as binding of viral nucleoprotein complexes, targeting to and modification of plasmodesmata, and release of the cargo after cell-to-cell transfer. For Abutilon mosaic virus (AbMV), phosphorylation of MP expressed in bacteria, yeast, and Nicotiana benthamiana plants, respectively, has been demonstrated in previous studies. Three phosphorylation sites (T221, S223, and S250) were identified in its C-terminal oligomerization domain by mass spectrometry, suggesting a regulation of MP by posttranslational modification. To examine the influence of the three sites on the self-interaction in more detail, MP mutants were tested for their interaction in yeast by two-hybrid assays, or by Förster resonance energy transfer (FRET) techniques in planta. Expression constructs with point mutations leading to simultaneous (triple) exchange of T221, S223, and S250 to either uncharged alanine (MPAAA), or phosphorylation charge-mimicking aspartate residues (MPDDD) were compared. MPDDD interfered with MP-MP binding in contrast to MPAAA. The roles of the phosphorylation sites for the viral life cycle were studied further, using plant-infectious AbMV DNA-B variants with the same triple mutants each. When co-inoculated with wild-type DNA-A, both mutants infected N. benthamiana plants systemically, but were unable to do so for some other plant species of the families Solanaceae or Malvaceae. Systemically infected plants developed symptoms and viral DNA levels different from those of wild-type AbMV for most virus-plant combinations. The results indicate a regulation of diverse MP functions by posttranslational modifications and underscore their biological relevance for a complex host plant-geminivirus interaction.