To bind or not to bind: Characterization of binding interactions between X29 and U8snoRNA [abstract]

Abstract only availableFaculty Mentor: Brenda Peculis, BiochemistryU8-snoRNP is involved in the processing of the 5.8S and the 28S rRNA, both of which are needed for the formation of the large ribosomal subunit (Peculis and Steitz, 1991). A nucleolar protein, dubbed X29, has the ability to bind and decap U8snoRNA, giving it the capacity to degrade U8RNA (Tomasevic and Peculis, 1999; Ghosh et al, 2004). Initially found in Xenopus, X29 is evolutionarily conserved in vertebrates from humans to sea squirts (MT, unpublished). The x-ray crystallography structure of X29 shows the protein can exist in the form of a homodimer (Scarsdale et al, 2006). The goal of this project was to determine whether the homodimer form or the monomeric version of X29 binds U8RNA and is catalytically active, as well as identify the protein:protein and protein:RNA contacts. I used chemical crosslinkers and identified a 60kD putative crosslink in X29, and formation of a 60kD band was also identified with the human homologue, H29K, but this forms with a lower efficiency. 4-thio-U mediated RNA (UV) crosslinking was used to identify the binding sites for X29 on U8RNA. All crosslinking assays were performed with mutant or truncated RNAs and proteins to more precisely map sites of interaction. The data from X29 were compared to that of H29K to determine whether the protein's activities were conserved among or differed between species. The results of these experiments showed that X29 and H29K both show abilities to form dimers and crosslink to U8 though to differing efficiencies. The U8 mutants have identified putative interaction sites between U8RNA and the proteins, which we are in the process of mapping more precisely

Binding properties of X29 protein and RNA

Abstract only availableThe Peculis Lab previously demonstrated that X29 protein binds U8 snoRNA with high affinity and specificity in Xenopus Laevis [1]. U8 snoRNA is a C/D box RNP required for pre-rRNA maturation of 5.8s and 28s rRNA in the nucleolus. X29 is a Nudix hydrolase that decaps the U8 RNA at the m7G and m227G caps [2]. Together these two components may interact in vivo to regulate the rate of ribosome biogenesis and thus, the rates of cell growth and cell division. My work has focused on characterizing the interaction between these two molecules and I have been working on two interrelated projects. The first project involves generating mutations to alter one amino acid in the protein sequence. Based on crystal structure data, the amino acid tryptophan, at position F49, lies in the putative RNA binding site on the X29 protein. The mutation will substitute a tryptophan for the 'wild type' phenylalanine residue at this position. After the mutagenesis, the protein is expressed in bacteria in BL21 cells and the mutated protein is purified. We predict the protein would contain a fluorescence property such that when analyzed by flourimitry we will be able to determine RNA binding and possibly address stoichiometry and dimer formation. The second project involves cross-linking wild type X29 protein to U8 RNA and mapping the cross-link on the RNA. Cross-linking reactions followed by reverse transcription using 5' end-labeled oligo DNA primers will identify 'stops' which are UV- and protein-dependent. We will be able to map the precise nucleotide on the RNA and interpret this in the framework of the proposed secondary structure for U8 snoRNA. The results of these experiments will greatly aid the research of the X29 protein and its binding capabilities to RNA. References: 1. Tomasevic, N. And B. Peculis, Identification of a U8 snoRNA-specific binding protein. J Biol Chem, 1999. 274: p. 35914-20. 2. Ghosh, T., et al., Xenopus U8 snoRNA binding protein is a conserved nuclear decapping enzyme. Mol Cell, 2004. 13: p. 817- 828.NSF-REU Program in Biological Sciences & Biochemistr

Exploring the functional decapping ability of the dogfish shark Nudt16 homolog

Abstract only availableA nuclear decapping and binding protein, X29/Nudt16, was originally characterized in Xenopus, and has also been characterized in humans. It is suggested that not only the protein's sequence, but also its functions has been preserved through evolution. The purpose of this study is to characterize a homologue of Nudt16 in the dogfish shark, which on an evolutionary scale is quite diverged from frogs and mammals. Here the open reading frame encoding the dogfish shark protein was amplified via PCR and cloned into an expression vector. When placed into bacteria under the proper growth conditions, a Histidine-tagged dogfish shark protein is synthesized. The protein can be purified from bacteria. The purified dogfish shark protein will be tested to determine if it has the same biochemical properties as the mammalian and frog proteins: can the protein decap RNA in vitro and can it bind RNA directly. Does this protein have catalytic activity that is essential for a healthy life?NSF grant to B. Peculus, NSF-REU Program in Biological Sciences & Biochemistr

Case Report : Micro-RNAs in Plasma From Bilateral Inferior Petrosal Sinus Sampling and Peripheral Blood From Corticotroph Pituitary Neuroendocrine Tumors

Funding Information: The authors acknowledge the Latvian Biomedical Research and Study Centre and the Genome Database of the Latvian Population for providing infrastructure, biological material and data. Funding Information: This research was funded by the European Regional Development Fund within the project RNA molecular determinants in development of pituitary adenoma” (1.1.1.1/18/A/089). Publisher Copyright: Copyright © 2022 Niedra, Peculis, Konrade, Balcere, Romanovs, Steina, Stukens, Sokolovska, Klovins and Rovite.Objective: Circulating miRNAs are found in bodily fluids including plasma and can serve as biomarkers for diseases. The aim of this study was to provide the first insight into the landscape of circulating miRNAs in close proximity to the adrenocorticotropic hormone (ACTH) secreting PitNET. To achieve this objective next-generation sequencing of miRNAs in plasma from bilateral inferior petrosal sinus sampling (BIPSS) - a gold standard in diagnosing ACTH-secreting PitNETs was carried out and selected miRNA candidates were further tested by RT-qPCR in independent patient cohorts. Methods: Sinistral (left) and dextral (right) BIPSS blood samples of the patient were collected in three time points: before the administration of corticotropin-releasing hormone, 5 and 15 minutes after stimulation. In differential expression analysis, sinistral plasma was compared with dextral. The selected miRNA candidates were tested in plasma by RT-qPCR in two patient groups: 1) in five ACTH secreting PitNET patients with plasma samples taken before and 24 hours after surgery, 2) in 12 ACTH secreting PitNET patients vs. 9 non-functioning PitNET patients. Results: BIPSS concluded that the highest amount of ACTH was released in the sinistral side at the 5th minute mark indicating a presence of a tumor. The highest amount of differentially expressed miRNAs was observed 5 minutes after stimulation (20 upregulated, 14 downregulated). At the 5th minute mark in sinistral plasma, two miRNAs were identified: hsa-miR-7-5p and hsa-miR-375-3p that were highly upregulated compared to other BIPSS samples and peripheral plasma samples. Further testing by qPCR revealed significant reduction of miR-7-5p in plasma 24 hours after surgery and upregulation in plasma of ACTH secreting PitNET patients compared to non-functioning PitNET patients (P =0.0013). Conclusions: By stimulating the ACTH secreting PitNET with CRH a rapid increase of two miRNAs (hsa-mir-7-5p, hsa-mir-375-3p) and ACTH can be observed in sinistral inferior petrosal (tumor side). A decrease of miR-7-5p in plasma after surgery and upregulation in plasma of ACTH secreting PitNET patients was discovered implying that further studies of this miRNA as diagnostic marker is needed.publishersversionPeer reviewe

Novel susceptibility loci identified in a genome-wide association study of type 2 diabetes complications in population of Latvia

Funding Information: The study was supported by European Regional Development Fund (ERDF) On Implementation of Activity 1.1.1.2 “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1 “To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure” of the Operational Programme “Growth and Employment”. Project No 1.1.1.2/VIAA/2/18/287 “Identification of clinical subgroups of Type 2 diabetes mellitus and application of pharmacogenetics in the development of personalized antidiabetic therapy”. The funding body was not involved in the design of the study, collection, analysis or interpretation of data, or writing the manuscript. Publisher Copyright: © 2021, The Author(s).Background: Type 2 diabetes complications cause a serious emotional and economical burden to patients and healthcare systems globally. Management of both acute and chronic complications of diabetes, which dramatically impair the quality of patients' life, is still an unsolved issue in diabetes care, suggesting a need for early identification of individuals with high risk for developing diabetes complications. Methods: We performed a genome-wide association study in 601 type 2 diabetes patients after stratifying them according to the presence or absence of four types of diabetes complications: diabetic neuropathy, diabetic nephropathy, macrovascular complications, and ophthalmic complications. Results: The analysis revealed ten novel associations showing genome-wide significance, including rs1132787 (GYPA, OR = 2.71; 95% CI = 2.02–3.64) and diabetic neuropathy, rs2477088 (PDE4DIP, OR = 2.50; 95% CI = 1.87–3.34), rs4852954 (NAT8, OR = 2.27; 95% CI = 2.71–3.01), rs6032 (F5, OR = 2.12; 95% CI = 1.63–2.77), rs6935464 (RPS6KA2, OR = 2.25; 95% CI = 6.69–3.01) and macrovascular complications, rs3095447 (CCDC146, OR = 2.18; 95% CI = 1.66–2.87) and ophthalmic complications. By applying the targeted approach of previously reported susceptibility loci we managed to replicate three associations: MAPK14 (rs3761980, rs80028505) and diabetic neuropathy, APOL1 (rs136161) and diabetic nephropathy. Conclusions: Together these results provide further evidence for the implication of genetic factors in the development of type 2 diabetes complications and highlight several potential key loci, able to modify the risk of developing these conditions. Moreover, the candidate variant approach proves a strong and consistent effect for multiple variants across different populations.publishersversionPeer reviewe

Whole exome sequencing reveals novel risk genes of pituitary neuroendocrine tumors

Funding Information: This research was supported by the European Regional Development Fund (ERDF), Measure 1.1.1.1 “Industry-Driven Research” project „Molecular markers of pituitary tumour development, progression and therapy response” (Project No. 1.1.1.1/16/A/066, 2017). The authors acknowledge the Latvian Biomedical Research and Study Centre and the Genome Database of the Latvian Population for providing infrastructure, biological material and data. Publisher Copyright: © 2022 Peculis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Somatic genetic alterations in pituitary neuroendocrine tumors (PitNET) tissues have been identified in several studies, but detection of overlapping somatic PitNET candidate genes is rare. We sequenced and by employing multiple data analysis methods studied the exomes of 15 PitNET patients to improve discovery of novel factors involved in PitNET development. PitNET patients were recruited to the study before PitNET removal surgery. For each patient, two samples for DNA extraction were acquired: venous blood and PitNET tissue. Exome sequencing was performed using Illumina NexSeq 500 sequencer and data analyzed using two separate workflows and variant calling algorithms: GATK and Strelka2. A combination of two data analysis pipelines discovered 144 PitNET specific somatic variants (mean = 9.6, range 0–19 per PitNET) of which all were SNVs. Also, we detected previously known GNAS PitNET mutation and identified somatic variants in 11 genes, which have contained somatic variants in previous WES and WGS studies of PitNETs. Noteworthy, this is the third study detecting somatic variants in gene RYR1 in the exomes of PitNETs. In conclusion, we have identified two novel PitNET candidate genes (AC002519.6 and AHNAK) with recurrent somatic variants in our PitNET cohort and found 13 genes overlapping from previous PitNET studies that contain somatic variants. Our study demonstrated that the use of multiple sequencing data analysis pipelines can provide more accurate identification of somatic variants in PitNETs.publishersversionPeer reviewe

A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei

Tissue-specific patterns of methylated deoxycytidine residues in the mammalian genome are preserved by postreplicative methylation of newly synthesized DNA. DNA methyltransferase (MTase) is here shown to associate with replication foci during S phase but to display a diffuse nucleoplasmic distribution in non-S phase cells. Analysis of DNA MTase-β-galactosidase fusion proteins has shown that association with replication foci is mediated by a novel targeting sequence located near the N-terminus of DNA MTase. This sequence has the properties expected of a targeting sequence in that it is not required for enzymatic activity, prevents proper targeting when deleted, and, when fused to β-galactosidase, causes the fusion protein to associate with replication foci in a cell cycle-dependent manner

Polymorphisms in MEN1 and DRD2 genes are associated with the occurrence and characteristics of pituitary adenomas

Publisher Copyright: © 2016 European Society of Endocrinology Published by Bioscientifica Ltd. Copyright: Copyright 2017 Elsevier B.V., All rights reserved.Objective: Although pituitary adenomas (PAs) affect a significant proportion of the population, only a fraction have the potential to become clinically relevant during an individual's lifetime, causing hormonal imbalance or complications due to mass effect. The overwhelming majority of cases are sporadic and without a clear familial history, and the genotype-phenotype correlation in PA patients is poorly understood. Our aim was to investigate the involvement of genes known for their role in familial cases on drug response and tumor suppression in the development and pathology of PAs in a patient group from Latvia. Design: The study included 143 cases and 354 controls, we investigated the role of single-nucleotide polymorphisms (SNPs) in seven genes (SSTR2, SSTR5, DRD2, MEN1, AIP, GNAS, and PRKAR1A) associated with pituitary tumor occurrence, phenotype, and clinical symptoms. Methods: Genotyping of 96 tag and nonsynonymous SNPs was performed in the genomic regions of interest. Results: We discovered a significant association (OR = 17.8, CI 0.95 = 2.18-145.5, P = 0.0002) between a rare MEN1 mutation (rs2959656) and clinically active adenoma in our patients. Additionally, rs7131056 at DRD2 was associated with a higher occurrence of extrasellar growth in patients with prolactinoma and somatotropinoma (OR = 2.79, CI 0.95 = 1.58-4.95, P = 0.0004). Conclusions: rs2959656, a nonsynonymous variant in MEN1, is associated with the development of clinically active PA. Furthermore, rs7131056 in DRD2 contributes to either faster growth of the adenoma or reduced symptomatic presentation, allowing PAs to become larger before detection.publishersversionPeer reviewe