Analyses and web interfaces for protein subcellular localization and gene expression data

Cataloged from PDF version of article.In order to benefit maximally from large scale molecular biology data generated by recent developments, it is important to proceed in an organized manner by developing databases, interfaces, data visualization and data interpretation tools. Protein subcellular localization and microarray gene expression are two of such fields that require immense computational effort before being used as a roadmap for the experimental biologist. Protein subcellular localization is important for elucidating protein function. We developed an automatically updated searchable and downloadable system called model organisms proteome subcellular localization database (MEP2SL) that hosts predicted localizations and known experimental localizations for nine eukaryotes. MEP2SL localizations highly correlated with high throughput localization experiments in yeast and were shown to have superior accuracies when compared with four other localization prediction tools based on two different datasets. Hence, MEP2SL system may serve as a reference source for protein subcellular localization information with its interface that provides various search and download options together with links and utilities for further annotations. Microarray gene expression technology enables monitoring of whole genome simultaneously. We developed an online installable searchable open source system called differentially expressed genes (DEG) that includes analysis and retrieval interfaces for Affymetrix HG-U133 Plus 2.0 arrays. DEG provides permanent data storage capabilities with its integration into a database and being an installable online tool and is valuable for groups who are not willing to submit their data on public servers.Bilen, BiterM.S

Development of methods for the analysis of deep sequencing data; applications to the discovery of functions of RNA-binding proteins

With the recent advances in nucleotide sequencing technologies, it became easy to generate tens of millions of reads with genome- or transcriptome-wide distribution with reduced cost and high accuracy. One of the applications of deep sequencing is the determination of the repertoire of targets of RNA-binding proteins. The method, called CLIP (for UV crosslinking and immune-precipitation) is now widely used to characterize a variety of proteins with regulatory as well as enzymatic functions. Here we focus on the statistical analysis of data obtained through a variant of CLIP, called PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced CLIP), which was applied to three different RNA binding proteins whose function was previously not well characterized: PAPD5 (PAP associated domain containing 5), DIS3L2 (DIS3 mitotic control homolog (S. cerevisiae)-like 2), and EWSR1 (Ewing sarcoma breakpoint region 1). Our computational analysis was instrumental for the definition of the main in vivo substrates of these proteins, which were confirmed by additional experiments. In the analysis, we also used extensively publicly available high-throughput data sets that enabled us make inferences about the function of the proteins. The main results of biological significance were as follows. We determined ribosomal RNAs are the main targets of PAPD5 and that the main substrates of the DIS3L2 nuclease are tRNAs and found that the tRNA-derived fragments processed by DIS3L2 could be loaded in the RNA silencing complex and be involved in gene silencing. Finally, we determined that EWSR1preferentially binds to RNAs that originate from instability-prone regions like sub-telomeres, known to be hotspots of genomic rearrangements, as well as other genes located in internal regions of chromosomes, that have been implicated in genomic translocations. These include EWSR1’s own pre-mRNA. All together this dissertation illustrates the point that when coupled with proper statistical analysis, CLIP is able to reveal targets of RNA-binding proteins that were difficult to study with other methods and that and integration of public domain datasets is very powerful in deciphering complex RNA-protein and regulatory RNA networks implicated in post-transcriptional gene regulation

Argonaute CLIP - A method to identify in vivo targets of miRNAs

microRNAs are important regulators of gene expression that guide translational repression and degradation of target mRNAs. Only relatively few miRNA targets have been characterized, and computational prediction is hampered by the relatively small number of nucleotides that seem to be involved in target recognition. Argonaute (Ago) crosslinking and immunoprecipitation (CLIP) in combination with next-generation sequencing proved to be a successful method for identifying targets of endogenous cellular miRNAs on a transcriptome-wide scale. Here we review various approaches to Ago CLIP, describe in detail the PAR-CLIP method and provide an outline of the necessary computational analysis for identification of in vivo miRNA binding sites

Analysis of CDS-located miRNA target sites suggests that they can effectively inhibit translation

Most of what is presently known about how miRNAs regulate gene expression comes from studies that characterized the regulatory effect of miRNA binding sites located in the 3' untranslated regions (UTR) of mRNAs. In recent years, there has been increasing evidence that miRNAs also bind in the coding region (CDS), but the implication of these interactions remains obscure because they have a smaller impact on mRNA stability compared with miRNA-target interactions that involve 3' UTRs. Here we show that miRNA-complementary sites that are located in both CDS and 3'-UTRs are under selection pressure and share the same sequence and structure properties. Analyzing recently published data of ribosome-protected fragment profiles upon miRNA transfection from the perspective of the location of miRNA-complementary sites, we find that sites located in the CDS are most potent in inhibiting translation, while sites located in the 3' UTR are more efficient at triggering mRNA degradation. Our study suggests that miRNAs may combine targeting of CDS and 3' UTR to flexibly tune the time scale and magnitude of their post-transcriptional regulatory effects

Peripheral T-Cells, B-Cells, and Monocytes from Multiple Sclerosis Patients Supplemented with High-Dose Vitamin D Show Distinct Changes in Gene Expression Profiles

Vitamin D is a steroid hormone that has been widely studied as a potential therapy for multiple sclerosis and other inflammatory disorders. Pre-clinical studies have implicated vitamin D in the transcription of thousands of genes, but its influence may vary by cell type. A handful of clinical studies have failed to identify an in vivo gene expression signature when using bulk analysis of all peripheral immune cells. We hypothesized that vitamin D’s gene signature would vary by immune cell type, requiring the analysis of distinct cell types. Multiple sclerosis patients (n = 18) were given high-dose vitamin D (10,400 IU/day) for six months as part of a prospective clinical trial (NCT01024777). We collected peripheral blood mononuclear cells from participants at baseline and again after six months of treatment. We used flow cytometry to isolate three immune cell types (CD4+ T-cells, CD19+ B-cells, CD14+ monocytes) for RNA microarray analysis and compared the expression profiles between baseline and six months. We identified distinct sets of differentially expressed genes and enriched pathways between baseline and six months for each cell type. Vitamin D’s in vivo gene expression profile in the immune system likely differs by cell type. Future clinical studies should consider techniques that allow for a similar cell-type resolution

Genome-Wide Transcriptional Reorganization Associated with Senescence-to-Immortality Switch during Human Hepatocellular Carcinogenesis

Senescence is a permanent proliferation arrest in response to cell stress such as DNA damage. It contributes strongly to tissue aging and serves as a major barrier against tumor development. Most tumor cells are believed to bypass the senescence barrier (become "immortal") by inactivating growth control genes such as TP53 and CDKN2A. They also reactivate telomerase reverse transcriptase. Senescence-to-immortality transition is accompanied by major phenotypic and biochemical changes mediated by genome-wide transcriptional modifications. This appears to happen during hepatocellular carcinoma (HCC) development in patients with liver cirrhosis, however, the accompanying transcriptional changes are virtually unknown. We investigated genome-wide transcriptional changes related to the senescence-to-immortality switch during hepatocellular carcinogenesis. Initially, we performed transcriptome analysis of senescent and immortal clones of Huh7 HCC cell line, and identified genes with significant differential expression to establish a senescence-related gene list. Through the analysis of senescence-related gene expression in different liver tissues we showed that cirrhosis and HCC display expression patterns compatible with senescent and immortal phenotypes, respectively; dysplasia being a transitional state. Gene set enrichment analysis revealed that cirrhosis/senescence-associated genes were preferentially expressed in non-tumor tissues, less malignant tumors, and differentiated or senescent cells. In contrast, HCC/immortality genes were up-regulated in tumor tissues, or more malignant tumors and progenitor cells. In HCC tumors and immortal cells genes involved in DNA repair, cell cycle, telomere extension and branched chain amino acid metabolism were up-regulated, whereas genes involved in cell signaling, as well as in drug, lipid, retinoid and glycolytic metabolism were down-regulated. Based on these distinctive gene expression features we developed a 15-gene hepatocellular immortality signature test that discriminated HCC from cirrhosis with high accuracy. Our findings demonstrate that senescence bypass plays a central role in hepatocellular carcinogenesis engendering systematic changes in the transcription of genes regulating DNA repair, proliferation, differentiation and metabolism

A single-cell transcriptomic atlas characterizes ageing tissues in the mouse

Ageing is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death1. Despite rapid advances over recent years, many of the molecular and cellular processes that underlie the progressive loss of healthy physiology are poorly understood2. To gain a better insight into these processes, here we generate a single-cell transcriptomic atlas across the lifespan of Mus musculus that includes data from 23 tissues and organs. We found cell-specific changes occurring across multiple cell types and organs, as well as age-related changes in the cellular composition of different organs. Using single-cell transcriptomic data, we assessed cell-type-specific manifestations of different hallmarks of ageing-such as senescence3, genomic instability4 and changes in the immune system2. This transcriptomic atlas-which we denote Tabula Muris Senis, or 'Mouse Ageing Cell Atlas'-provides molecular information about how the most important hallmarks of ageing are reflected in a broad range of tissues and cell types