HnRNPK maintains single strand RNA through controlling double-strand RNA in mammalian cells

Although antisense transcription is a widespread event in the mammalian genome, double-stranded RNA (dsRNA) formation between sense and antisense transcripts is very rare and mechanisms that control dsRNA remain unknown. By characterizing the FGF-2 regulated transcriptome in normal and cancer cells, we identified sense and antisense transcripts IER3 and IER3-AS1 that play a critical role in FGF-2 controlled oncogenic pathways. We show that IER3 and IER3-AS1 regulate each other\u27s transcription through HnRNPK-mediated post-transcriptional regulation. HnRNPK controls the mRNA stability and colocalization of IER3 and IER3-AS1. HnRNPK interaction with IER3 and IER3-AS1 determines their oncogenic functions by maintaining them in a single-stranded form. hnRNPK depletion neutralizes their oncogenic functions through promoting dsRNA formation and cytoplasmic accumulation. Intriguingly, hnRNPK loss-of-function and gain-of-function experiments reveal its role in maintaining global single- and double-stranded RNA. Thus, our data unveil the critical role of HnRNPK in maintaining single-stranded RNAs and their physiological functions by blocking RNA-RNA interactions

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Chromatin and transcriptome-based integrative approaches to profile functional long noncoding RNAs - A computational approach

One of the major hallmarks of cancer is aberrant or uncontrollable cell division, which occurs due to a defective cell cycle process. During the synthesis phase (S-phase) of the cell cycle, before cell division or mitosis phase, the DNA in the cell makes a new copy to pass on genetic information to the daughter cells. Therefore, S-phase is one of the crucial steps for a successful cell division to occur. The DNA in the nucleus is wrapped around a set of proteins called histones, forming nucleosomes, and multiple nucleosomes give rise to the higher-order chromatin structure. This well-established chromatin structure determines which portion of DNA or gene gets activated or suppressed by switching between open or closed chromatin states. Tri- or di-methylation of lysine 4 from histone 3 (H3K4me2/3) leads to open chromatin, which in turn promotes active gene transcription. The product of gene transcription is either protein-coding mRNA that translates into protein for its function or noncoding RNA, which do not code for any protein and function as RNA. However, the human genome project has identified that protein-coding genes only constitute 2% of the genome, and the vast majority of it is noncoding. Unlike protein-coding genes, the significance of RNAs transcribed from the noncoding genome is not well-established. Apart from housekeeping noncoding RNAs (rRNA, tRNA, snRNA, and snoRNA) and microRNAs (miRNAs), most functional noncoding RNAs fall into the long noncoding RNA (lncRNA) category. In this thesis, we implemented comprehensive computational approaches to identify functionally relevant lncRNAs by analyzing chromatin and transcriptome-based sequencing datasets. In the first study (paper I), using a transcriptome approach, we profiled lncRNAs associated with the S-phase stage of the cell cycle. We demonstrated the oncogenic properties of various S-phase associated lncRNAs in multiple cancers. Earlier, studies proposed that chromatin-associated RNAs, with the help of chromatin-modifying enzymes, determines the active/open or close chromatin status to promote or suppress gene transcription. Hence, in the second study (paper II), we used chromatin-based approaches to propose a possible mechanism through which the active chromatin- associated lncRNAs may function. We show that active chromatin-associated lncRNAs regulate their partner genes in-cis by recruiting the WDR5 chromatin modifier to establish an open chromatin structure at the partner protein-coding gene promoters. In our third study (paper III), we integrated both transcriptome and chromatin-based approaches to find early development-associated lncRNAs. Here, we focused on tracing the molecular footprints of sperm lncRNAs throughout the stages of organismal development. For this purpose, we integrated datasets from gametes, preimplantation and post-implantation stages of an embryo. Interestingly, we observed distinct chromatin structures in the sperm. Also, sperm lncRNAs were active during the onset of zygotic genome activation in the preimplantation stages and in cancers. In summary, this study reveals a unique set of sperm-specific lncRNAs that are temporally activated during preimplantation stages and also aberrantly expressed in multiple cancers. Overall, the present thesis provides an extensive catalogue of functionally relevant lncRNAs that can take part in cell cycle regulation, cancer, chromatin modulation, and organism development. Our studies can serve as a comprehensive resource for future investigations on lncRNAs

Chromatin and transcriptome-based integrative approaches to profile functional long noncoding RNAs - A computational approach

One of the major hallmarks of cancer is aberrant or uncontrollable cell division, which occurs due to a defective cell cycle process. During the synthesis phase (S-phase) of the cell cycle, before cell division or mitosis phase, the DNA in the cell makes a new copy to pass on genetic information to the daughter cells. Therefore, S-phase is one of the crucial steps for a successful cell division to occur. The DNA in the nucleus is wrapped around a set of proteins called histones, forming nucleosomes, and multiple nucleosomes give rise to the higher-order chromatin structure. This well-established chromatin structure determines which portion of DNA or gene gets activated or suppressed by switching between open or closed chromatin states. Tri- or di-methylation of lysine 4 from histone 3 (H3K4me2/3) leads to open chromatin, which in turn promotes active gene transcription. The product of gene transcription is either protein-coding mRNA that translates into protein for its function or noncoding RNA, which do not code for any protein and function as RNA. However, the human genome project has identified that protein-coding genes only constitute 2% of the genome, and the vast majority of it is noncoding. Unlike protein-coding genes, the significance of RNAs transcribed from the noncoding genome is not well-established. Apart from housekeeping noncoding RNAs (rRNA, tRNA, snRNA, and snoRNA) and microRNAs (miRNAs), most functional noncoding RNAs fall into the long noncoding RNA (lncRNA) category. In this thesis, we implemented comprehensive computational approaches to identify functionally relevant lncRNAs by analyzing chromatin and transcriptome-based sequencing datasets. In the first study (paper I), using a transcriptome approach, we profiled lncRNAs associated with the S-phase stage of the cell cycle. We demonstrated the oncogenic properties of various S-phase associated lncRNAs in multiple cancers. Earlier, studies proposed that chromatin-associated RNAs, with the help of chromatin-modifying enzymes, determines the active/open or close chromatin status to promote or suppress gene transcription. Hence, in the second study (paper II), we used chromatin-based approaches to propose a possible mechanism through which the active chromatin- associated lncRNAs may function. We show that active chromatin-associated lncRNAs regulate their partner genes in-cis by recruiting the WDR5 chromatin modifier to establish an open chromatin structure at the partner protein-coding gene promoters. In our third study (paper III), we integrated both transcriptome and chromatin-based approaches to find early development-associated lncRNAs. Here, we focused on tracing the molecular footprints of sperm lncRNAs throughout the stages of organismal development. For this purpose, we integrated datasets from gametes, preimplantation and post-implantation stages of an embryo. Interestingly, we observed distinct chromatin structures in the sperm. Also, sperm lncRNAs were active during the onset of zygotic genome activation in the preimplantation stages and in cancers. In summary, this study reveals a unique set of sperm-specific lncRNAs that are temporally activated during preimplantation stages and also aberrantly expressed in multiple cancers. Overall, the present thesis provides an extensive catalogue of functionally relevant lncRNAs that can take part in cell cycle regulation, cancer, chromatin modulation, and organism development. Our studies can serve as a comprehensive resource for future investigations on lncRNAs

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol

Additional file 9: of GeneSCF: a real-time based functional enrichment tool with support for multiple organisms

Functional enrichment results from GeneSCF for p53 bound genes at 12 h (Case study 2) predicted by GeneSCF using Gene ontology, Molecular Function (GO_MF) as a reference database. (XLS 97 kb

Additional file 1: of GeneSCF: a real-time based functional enrichment tool with support for multiple organisms

The list of organisms supported by GeneSCF for KEGG pathway database. (XLS 542 kb

Chromatin RNA Immunoprecipitation (ChRIP)

Researchers have recently had a growing interest in understanding the functional role of long noncoding RNAs (lncRNAs) in chromatin organization. Accumulated evidence suggests lncRNAs could act as interphase molecules between chromatin and chromatin remodelers to define the epigenetic code. However, it is not clear how lncRNAs target chromatin remodelers to specific chromosomal regions in order to establish a functionally distinct epigenetic state of chromatin. We developed and optimized chromatin RNA immunoprecipitation (ChRIP) technology to characterize the lncRNAs associated with active and inactive chromatin compartments. Use of ChRIP to identify chromatin-bound lncRNA will further improve our knowledge regarding the functional role of lncRNAs in establishing epigenetic modifications of chromatin