Williams-Beureni sündroomi kromosoomiregiooni valk WBSCR22 kui ribosoomi biogeneesifaktor

Väitekirja elektrooniline versioon ei sisalda publikatsiooneRibosoomide biogeneesil osaleb ligi 300 valku, millest ligi kolmandik on seotud genoomsete haiguste ja kasvajate tekkega. Williams-Beureni sündroom (WBS) on arenguhäire, mida põhjustab umbes 30 geeni sisaldava regiooni puudumine seitsmendast kromosoomist. WBS patsientidel esineb rida erivaid probleeme – südame-veresoonkonna haigused, sidekoe arenguhäired, neuroloogilised probleemid, spetsiifiline kognitiivne profiil, kasvuprobleemid ja iseloomulikud näojooned. Ümberkorraldused WBS kromosoomiregioonis tekivad madalakoopiaarvuliste kordusjärjestuste esinemise tõttu, kuid hetkel pole teada, kuidas üksikute geenide koopiaarvu muutus põhjustab haigustunnuseid. Arvatakse, et paljud geenid on olulised WBS-i tekkel, mistõttu on vajalik nendelt geenidelt ekspresseeritavate valkude funktsioonide uurimine. Üheks WBS lookusest avalduvaks valguks, mille funktsioon enne antud töö raames sooritatud katsete tegemist oli teadmata, on WBSCR22. WBSCR22 iseloomustamine on oluline ka seetõttu, et tema avaldumistase on tõusnud mitmete kasvajate korral ja on teada, et ta mõjutab kasvajarakkude elulemust ja metastaaside teket. Antud doktoritöö esimese osa eesmärgiks oli WBSCR22 funktsiooni uurimine imetaja rakkudes. Leiti, et WBSCR22 valk asub hajusalt rakutuumas, kuid esineb ka valgu kogunemist tuumakesse – piirkonda, kus toimub ribosomaalse RNA (rRNA) süntees ja algab ribosoomide biogenees. Doktoritöös leiti, et WBSCR22 on oluline rakkude kasvuks, ribosoomi väikese subühiku biogeneesiks ja pre-rRNA protsessinguks. Täpsemalt, WBSCR22 ekspressiooni allareguleerimine põhjustab küpse 18S rRNA viimase eellase, 18S-E pre-rRNA, kuhjumist raku tuumas. See toob kaasa 18S rRNA ja ribosoomi väikese subühiku hulga vähenemise tsütoplasmas, mis võib viia rakkude aeglase jagunemiseni. Doktoritöös leiti, et üheks WBSCR22-ga seonduvaks valguks on TRMT112. TRMT112 paiknemine rakus on määratud WBSCR22 poolt: WBSCR22-TRMT112 kompleks asub raku tuumas ning kompleksi teke on vajalik WBSCR22 stabiilsuseks. TRMT112 hulga vähenemisel suunatakse WBSCR22 lagundamisele. Doktoritöö teises osas analüüsiti retroviiruste Gag valgu poolt indutseeritud viiruslaadsete partiklite (VLP-de) valgulist koostist ning avastati, et nendes VLP-des esineb suur hulk rakulisi valke, sealhulgas ribosoomivalke, kuid nende roll VLP-des ei ole teada.In human cells, almost 300 trans-acting factors are required for the production of ribosomes. Investigating the functions of these proteins is of biomedical importance, as one third of the factors are related to genetic diseases and cancer. Williams-Beuren syndrome is a developmental disorder caused by the contiguous deletion of 26–28 genes from chromosome 7. This genomic disorder is characterized by cardiovascular abnormalities, connective tissue anomalies, a characteristic neurocognitive and behavioural profile, growth delay, and a subtle but distinctive facial dysmorphology. The low copy repeat blocks of the seventh chromosome predispose to genomic rearrangements. However, it is not known how the alterations in the copy number of individual genes contribute to the disease phenotypes. Several genes probably contribute to the phenotype and therefore, it is important to study the cellular functions of the proteins expressed from the WBS locus. The gene encoding for WBSCR22 protein is deleted in WBS, and studying the function of WBSCR22 is also relevant for cancer biology, since several works have demonstrated that WBSCR22 is upregulated in various types of cancers and regulates the survival and metastatic potential of cancer cells. The first part of this dissertation is focused on studying the function of the WBSCR22 protein in mammalian cells, and revealed that WBSCR22 is important for cell growth, ribosome biogenesis and pre-rRNA processing. More precisely, silencing of WBSCR22 expression causes the accumulation of 18S-E pre-rRNA, the final precursor of 18S rRNA, in the cell nucleus, leading to a reduced amount of mature 18S rRNA and ribosome small subunit. In addition, it was found that WBSCR22 interacts with TRMT112 protein and that this interaction is important for the stability of WBSCR22 protein. In conditions where the amount of TRMT112 is limited, WBSCR22 is degraded by the proteasome. The second part of this thesis is focused on analysing the protein content of virus-like particles (VLPs) induced by the expression of murine leukaemia virus (MLV) Gag protein. The results revealed that these VLPs contain various cellular proteins, including ribosomal proteins of both subunits

Precise, Genotype-First Breast Cancer Prevention : Experience With Transferring Monogenic Findings From a Population Biobank to the Clinical Setting

Although hereditary breast cancer screening and management are well accepted and established in clinical settings, these efforts result in the detection of only a fraction of genetic predisposition at the population level. Here, we describe our experience from a national pilot study (2018-2021) in which 180 female participants of Estonian biobank (of >150,000 participants in total) were re-contacted to discuss personalized clinical prevention measures based on their genetic predisposition defined by 11 breast cancer-related genes. Our results show that genetic risk variants are relatively common in the average-risk Estonian population. Seventy-five percent of breast cancer cases in at-risk subjects occurred before the age of 50 years. Only one-third of subjects would have been eligible for clinical screening according to the current criteria. The participants perceived the receipt of genetic risk information as valuable. Fluent cooperation of project teams supported by state-of-art data management, quality control, and secure transfer can enable the integration of research results to everyday medical practice in a highly efficient, timely, and well-accepted manner. The positive experience in this genotype-first breast cancer study confirms the value of using existing basic genomic data from population biobanks for precise prevention.Peer reviewe

Dissecting the polygenic basis of atherosclerosis via disease-associated cell state signatures

Coronary artery disease (CAD) is a pandemic disease where up to half of the risk is explained by genetic factors. Advanced insights into the genetic basis of CAD require deeper understanding of the contributions of different cell types, molecular pathways, and genes to disease heritability. Here, we investigate the biological diversity of atherosclerosis-associated cell states and interrogate their contribution to the genetic risk of CAD by using single-cell and bulk RNA sequencing (RNA-seq) of mouse and human lesions. We identified 12 disease-associated cell states that we characterized further by gene set functional profiling, ligand-receptor prediction, and transcription factor inference. Importantly, Vcam1+ smooth muscle cell state genes contributed most to SNP-based heritability of CAD. In line with this, genetic variants near smooth muscle cell state genes and regulatory elements explained the largest fraction of CAD-risk variance between individuals. Using this information for variant prioritization, we derived a hybrid polygenic risk score (PRS) that demonstrated improved performance over a classical PRS. Our results provide insights into the biological mechanisms associated with CAD risk, which could make a promising contribution to precision medicine and tailored therapeutic interventions in the future.publishedVersionPeer reviewe

Two new Neuratelia Rondani (Diptera, Mycetophilidae) species from Western Palaearctic: a case of limited congruence between morphology and DNA sequence data

Two new Mycetophilidae species, Neuratelia jabalmoussae sp. n. and Neuratelia salmelai sp. n. are described on the basis of material collected from Lebanon, Estonia and Finland. Detailed figures of male terminalia and photographs of general facies are provided along with discussions of their morphological distinction from sibling species. For the first time molecular characters are used to distinguish new fungus gnat species. Molecular analysis relies on cytochrome oxidase subunit one (COI) but has additionally been corroborated by information from the 28S and ITS2 regions of nuclear ribosomal DNA. Situations where morphological and molecular data provide conflicting evidence for species delimitation are discussed. A new country record from Georgia is provided for Neuratelia caucasica

The Stability of Ribosome Biogenesis Factor WBSCR22 Is Regulated by Interaction with TRMT112 via Ubiquitin-Proteasome Pathway.

The human WBSCR22 protein is a 18S rRNA methyltransferase involved in pre-rRNA processing and ribosome 40S subunit biogenesis. Recent studies have shown that the protein function in ribosome synthesis is independent of its enzymatic activity. In this work, we have studied the WBSCR22 protein interaction partners by SILAC-coupled co-immunoprecipitation assay and identified TRMT112 as the interaction partner of WBSCR22. Knock-down of TRMT112 expression decreased the WBSCR22 protein level in mammalian cells, suggesting that the stability of WBSCR22 is regulated through the interaction with TRMT112. The localization of the TRMT112 protein is determined by WBSCR22, and the WBSCR22-TRMT112 complex is localized in the cell nucleus. We provide evidence that the interaction between WBSCR22/Bud23 and TRMT112/Trm112 is conserved between mammals and yeast, suggesting that the function of TRMT112 as a co-activator of methyltransferases is evolutionarily conserved. Finally, we show that the transiently expressed WBSCR22 protein is ubiquitinated and degraded through the proteasome pathway, revealing the tight control of the WBSCR22 protein level in the cells

The Stability of Ribosome Biogenesis Factor WBSCR22 Is Regulated by Interaction with TRMT112 via Ubiquitin-Proteasome Pathway

<div><p>The human WBSCR22 protein is a 18S rRNA methyltransferase involved in pre-rRNA processing and ribosome 40S subunit biogenesis. Recent studies have shown that the protein function in ribosome synthesis is independent of its enzymatic activity. In this work, we have studied the WBSCR22 protein interaction partners by SILAC-coupled co-immunoprecipitation assay and identified TRMT112 as the interaction partner of WBSCR22. Knock-down of TRMT112 expression decreased the WBSCR22 protein level in mammalian cells, suggesting that the stability of WBSCR22 is regulated through the interaction with TRMT112. The localization of the TRMT112 protein is determined by WBSCR22, and the WBSCR22-TRMT112 complex is localized in the cell nucleus. We provide evidence that the interaction between WBSCR22/Bud23 and TRMT112/Trm112 is conserved between mammals and yeast, suggesting that the function of TRMT112 as a co-activator of methyltransferases is evolutionarily conserved. Finally, we show that the transiently expressed WBSCR22 protein is ubiquitinated and degraded through the proteasome pathway, revealing the tight control of the WBSCR22 protein level in the cells.</p></div

Expression of WBSCR22 mutants defective in TRMT112 binding.

<p>(A) Hela, U2OS, HepG2 and COS-7 cells were transfected with plasmids expressing EGFP, EGFP-WBSCR22, EGFP-WBSCR22-KT/AA and EGFP-WBSCR22-D117A proteins. The amount of EGFP positive cells was analyzed by flow cytometry 24, 48 and 72 hours after transfection. (B) Expression of TRMT112 and WBSCR22 proteins in HeLa cells. HeLa cells were transfected with plasmids encoding for wt WBSCR22 and its mutants with and without plasmid for TRMT112. Transfected cells were harvested 24 hours after electroporation, the lysate of 10⁵ cells was loaded on each lane and analyzed by western blot using antibody against E2Tag and α-tubulin. The non-specific signal is shown by asterisk. (C-E) Co-immunoprecipitation of WBSCR22 and TRMT112 proteins. HeLa (C) and COS-7 (E) cells were transfected with plasmids encoding for wt and mutant WBSCR22 proteins and lysed 24 hours later. (D) HeLa cells were transfected with plasmid encoding for E2Tag-TRMT112. In all cases, co-immunoprecipitation was performed using antibody against E2Tag and immunoblotting with antibodies against WBSCR22 and TRMT112. Proteins of the extract (Input; 10% of cell lysate) and pulled-down fraction (IP) were analyzed by immunoblotting.</p

Depletion of WBSCR22 reduces cell growth.

<p>(A) Protein expression of siWBSCR22 and siNeg. transfected cells was determined by western blot analysis using anti-WBSCR22 and anti-tubulin antibodies. Proteins from 10⁵ cells are loaded on each lane. (B) HeLa cells were transfected with siWBSCR22 or a control, siNeg, and the cell growth was monitored for five days. Average of three independent transfection experiments is shown.</p

The WBSCR22 protein in WBS patient lymhoblastoid cell lines.

<p>(A) Extracts from wild-type (E001 and E004) and three WBS patient-derived lymphoblastoid cell lines (GM13472, GM13473, GM13482) were immunoblotted to visualise the indicated proteins. (B) Quantification of western blot analysis. Average of three independent experiments with standard deviations is shown. The P value is in all cases below 0.005 using Student’s t-test.</p

WBSCR22 interacts with the TRMT112 protein in mammalian cells.

<p>(A) WBSCR22 protein expression in U2OS and U2OS cell line stably expressing the E2Tag-WBSCR22 was determined by western blot using anti-WBSCR22 antibody. α-tubulin detected with anti-α-tubulin antibodies is used as a loading control. (B) Experimental scheme of the SILAC coupled co-immunoprecipitation assay performed. (C) The WBSCR22 protein interaction partners in U2OS cells stably expressing the WBSCR22 protein. H/L ratio shows the relative enrichment of the peptides of proteins pulled-down with WBSCR22 compared to mock control. (D) The WBSCR22 protein interacts with TRMT112. The WBSCR22 protein was immunoprecipitated from U2OS and U2OS-E2Tag-WBSCR22 cell lysates with an antibody against E2Tag. Proteins of the extract (Input; 10% of the lysate and pulled-down fraction (IP) were analyzed by immunoblotting with antibodies against WBSCR22, TRMT112 and C1QBP.</p