The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression

Inner nuclear membrane proteins containing a LEM (LAP2, emerin, and MAN1) domain participate in different processes, including chromatin organization, gene expression, and nuclear envelope biogenesis. In this study, we identify a robust genetic interaction between transcription export (TREX) factors and yeast Src1, an integral inner nuclear membrane protein that is homologous to vertebrate LEM2. DNA macroarray analysis revealed that the expression of the phosphate-regulated genes PHO11, PHO12, and PHO84 is up-regulated in src1Δ cells. Notably, these PHO genes are located in subtelomeric regions of chromatin and exhibit a perinuclear location in vivo. Src1 spans the nuclear membrane twice and exposes its N and C domains with putative DNA-binding motifs to the nucleoplasm. Genome-wide chromatin immunoprecipitation–on-chip analyses indicated that Src1 is highly enriched at telomeres and subtelomeric regions of the yeast chromosomes. Our data show that the inner nuclear membrane protein Src1 functions at the interface between subtelomeric gene expression and TREX-dependent messenger RNA export through the nuclear pore complexes

Transmembrane protein PERP is a component of tessellate junctions and of other junctional and non-junctional plasma membrane regions in diverse epithelial and epithelium-derived cells

Protein PERP (p53 apoptosis effector related to PMP-22) is a small (21.4 kDa) transmembrane polypeptide with an amino acid sequence indicative of a tetraspanin character. It is enriched in the plasma membrane and apparently contributes to cell-cell contacts. Hitherto, it has been reported to be exclusively a component of desmosomes of some stratified epithelia. However, by using a series of newly generated mono- and polyclonal antibodies, we show that protein PERP is not only present in all kinds of stratified epithelia but also occurs in simple, columnar, complex and transitional epithelia, in various types of squamous metaplasia and epithelium-derived tumors, in diverse epithelium-derived cell cultures and in myocardial tissue. Immunofluorescence and immunoelectron microscopy allow us to localize PERP predominantly in small intradesmosomal locations and in variously sized, junction-like peri- and interdesmosomal regions (“tessellate junctions”), mostly in mosaic or amalgamated combinations with other molecules believed, to date, to be exclusive components of tight and adherens junctions. In the heart, PERP is a major component of the composite junctions of the intercalated disks connecting cardiomyocytes. Finally, protein PERP is a cobblestone-like general component of special plasma membrane regions such as the bile canaliculi of liver and subapical-to-lateral zones of diverse columnar epithelia and upper urothelial cell layers. We discuss possible organizational and architectonic functions of protein PERP and its potential value as an immunohistochemical diagnostic marker

A time-resolved proteomic and prognostic map of COVID-19

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease

Rare Drosha Splice Variants Are Deficient in MicroRNA Processing but Do Not Affect General MicroRNA Expression in Cancer Cells12

Drosha is a key enzyme in microRNA biogenesis, generating the precursor miRNA (pre-miRNA) by excising the stem-loop embedded in the primary transcripts (pri-miRNA). The specificity for the pri-miRNAs and determination of the cleavage site are provided by its binding partner DGCR8, which is necessary for efficient processing. The crucial Drosha domains for pri-miRNA cleavage are the middle part, the two enzymatic RNase III domains (RIIID), and the dsRNA binding domain (dsRBD) in the C-terminus. Here, we identify alternatively spliced transcripts in human melanoma and NT2 cell lines, encoding C-terminally truncated Drosha proteins lacking part of the RIIIDb and the entire dsRBD. Proteins generated from these alternative splice variants fail to bind to DGCR8 but still interact with Ewing sarcoma protein (EWS). In vitro as well as in vivo, the Drosha splice variants are deficient in pri-miRNA processing. However, the aberrant transcripts in melanoma cells do not consistently reduce mature miRNA levels compared with melanoma cell lines lacking those splice variants, possibly owing to their limited abundance. Our findings show that alternative processing-deficient Drosha splice variants exist in melanoma cells. In elevated amounts, these alternatively spliced transcripts could provide one potential mechanism accounting for the deregulation of miRNAs in cancer cells. On the basis of our results, the search for alternative inactive splice variants might be fruitful in different tumor entities to unravel the molecular basis of the previously observed decreased microRNA processing efficiency in cancer

(A and B) Relative increases (/wt, when wt) or decreases (wt/, when wt) of gene expression were plotted versus their distance to the closest telomere (A) or centromere (B)

A sliding window of 100 genes was used. The average of the 100 genes was used for the y axis, and the distance of the central gene in the window was used for the x axis. The bottom panel of A is an expanded view of the top graph showing the ∼27-kb region close to the telomeres, which exhibits a misregulation in the deletion strain. (C) Expression levels of mRNAs in wt and Δ cells. Total RNA of wt and Δ cells grown in HP and LP was prepared, and cDNA was analyzed by quantitative RT-PCR using specific primers for , , and . Each gene was assayed in triplicates. The mRNA levels of wt HP expression are set as one. One representative dataset of five times independently isolated RNA is shown. Error bars represent SD.<p><b>Copyright information:</b></p><p>Taken from "The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression"</p><p></p><p>The Journal of Cell Biology 2008;182(5):897-910.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528585.</p><p></p

(A) The double-disrupted strains were transformed with the respective plasmid-borne wt or mutant genes

Growth was analyzed by spotting transformants in 10-fold serial dilutions on 5-FOA–containing plates at the indicated temperature for 5 d or on synthetic dextrose complete–Leu-Trp for 3 d (Δ, , and ). No growth indicates synthetic lethality. (B) Schematic representation of the genetic network between and factors involved in transcription-coupled mRNA export. Arrows to gray components indicate synthetic lethality/enhancement, and proteins depicted in white are genetically not linked to .<p><b>Copyright information:</b></p><p>Taken from "The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression"</p><p></p><p>The Journal of Cell Biology 2008;182(5):897-910.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528585.</p><p></p

(A) Schematic overview of pre-mRNA, mRNA, and protein products upon alternative splicing

Either a 126- or a 130-nt intron can be excised by using two alternative 5′ splice sites. In the latter case, a frame shift results in an earlier stop codon and, therefore, in a shorter protein with a different amino acid sequence at the C terminus compared with Src1-L. Conserved domains (HEH/LEM and MSC) and transmembrane domains (M) are indicated. Numbers represent amino acid residues. (B) Whole cell lysates of N- (TAP-Src1) and C-terminal TAP-tagged Src1-L or Src1-S were analyzed by SDS-PAGE followed by Western blotting using anti-ProtA antibodies. (C) Genetic relationship of splice variants with TREX–THO and TREX-2 components. The double-disruption strains were transformed with empty vector, GFP-Src1 splice variants, and the respective TREX component. Transformants were spotted in 10-fold serial dilutions on 5-FOA–containing plates for 5 d at the indicated temperatures.<p><b>Copyright information:</b></p><p>Taken from "The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression"</p><p></p><p>The Journal of Cell Biology 2008;182(5):897-910.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528585.</p><p></p

The cancer-associated microprotein CASIMO1 controls cell proliferation and interacts with squalene epoxidase modulating lipid droplet formation

Breast cancer is a leading cause of cancer-related death in women. Small open reading frame (sORF)-encoded proteins or microproteins constitute a new class of molecules often transcribed from presumed long non-coding RNA transcripts (lncRNAs). The translation of some of these sORFs has been confirmed, but their cellular function and importance remains largely unknown. Here, we report the identification and characterization of a novel microprotein of 10 kDa, which we named Cancer-Associated Small Integral Membrane Open reading frame 1 (CASIMO1). CASIMO1 RNA is overexpressed predominantly in hormone receptor-positive breast tumors. Its knockdown leads to decreased proliferation in multiple breast cancer cell lines. Its loss disturbs the organization of the actin cytoskeleton, leads to inhibition of cell motility, and causes a G(0)/G(1) cell cycle arrest. The proliferation phenotype upon overexpression is observed only with CASIMO1 protein expression, but not with a non-translatable mutant attributing the effects to the sORF-derived protein rather than a lncRNA function. CASIMO1 microprotein interacts with squalene epoxidase (SQLE), a key enzyme in cholesterol synthesis and a known oncogene in breast cancer. Overexpression of CASIMO1 leads to SQLE protein accumulation without affecting its RNA levels and increased lipid droplet clustering, while knockdown of CASIMO1 decreased SQLE protein abundance and ERK phosphorylation downstream of SQLE. Importantly, SQLE knockdown mimicked the CASIMO1 knockdown phenotype and in turn SQLE overexpression fully rescued the effect of CASIMO1 knockdown. These findings establish CASIMO1 as the first functional microprotein that plays a role in carcinogenesis and is implicated in the cell lipid homeostasis