1.Use of Bru-Seq and BruChase-Seq for genome-wide assessment of the synthesis and stability of RNA.
Paulsen MT1, Veloso A2, Prasad J1, Bedi K3, Ljungman EA1, Magnuson B1, Wilson TE4, Ljungman M5. Methods. 2014 May 1;67(1):45-54. doi: 10.1016/j.ymeth.2013.08.015. Epub 2013 Aug 21.
Gene expression studies commonly examine total cellular RNA, which only provides information about its steady-state pool of RNA. It remains unclear whether differences in the steady-state reflects variable rates of transcription or RNA degradation. To specifically monitor RNA synthesis and degradation genome-wide, we developed Bru-Seq and BruChase-Seq. These assays are based on metabolic pulse-chase labeling of RNA using bromouridine (Bru). In Bru-Seq, recently labeled RNAs are sequenced to reveal spans of nascent transcription in the genome. In BruChase-Seq, cells are chased in uridine for different periods of time following Bru-labeling, allowing for the isolation of RNA populations of specific ages. Here we describe these methodologies in detail and highlight their usefulness in assessing RNA synthesis and stability as well as splicing kinetics with examples of specific genes from different human cell lines.
2.Nuclear structures in Tribolium castaneum oocytes.
Bogolyubov DS1, Batalova FM, Kiselyov AM, Stepanova IS. Cell Biol Int. 2013 Oct;37(10):1061-79. doi: 10.1002/cbin.10135. Epub 2013 Jun 24.
The first ultrastructural and immunomorphological characteristics of the karyosphere (karyosome) and extrachromosomal nuclear bodies in the red flour beetle, Tribolium castaneum, are presented. The karyosphere forms early in the diplotene stage of meiotic prophase by the gathering of all oocyte chromosomes in a limited nuclear volume. Using the BrUTP assay, T. castaneum oocyte chromosomes united in the karyosphere maintain their transcriptional activity until the end of oocyte growth. Hyperphosphorylated RNA polymerase II and basal transcription factors (TFIID and TFIIH) were detected in the perichromatin region of the karyosphere. The T. castaneum karyosphere has an extrachromosomal capsule that separates chromosomes from the rest of the nucleoplasm. Certain structural proteins (F-actin, lamin B) were found in the capsule. Unexpectedly, the karyosphere capsule in T. castaneum oocytes was found to be enriched in TMG-capped snRNAs, which suggests that the capsule is not only a structural support for the karyosphere, but may be involved in biogenesis of snRNPs.
3.Analysis of RNA decay factor mediated RNA stability contributions on RNA abundance.
Maekawa S1, Imamachi N2, Irie T3, Tani H4, Matsumoto K5, Mizutani R6, Imamura K7, Kakeda M8, Yada T9, Sugano S10, Suzuki Y11,12, Akimitsu N13. BMC Genomics. 2015 Mar 6;16:154. doi: 10.1186/s12864-015-1358-y.
BACKGROUND: Histone epigenome data determined by chromatin immunoprecipitation sequencing (ChIP-seq) is used in identifying transcript regions and estimating expression levels. However, this estimation does not always correlate with eventual RNA expression levels measured by RNA sequencing (RNA-seq). Part of the inconsistency may arise from the variance in RNA stability, where the transcripts that are more or less abundant than predicted RNA expression from histone epigenome data are inferred to be more or less stable. However, there is little systematic analysis to validate this assumption. Here, we used stability data of whole transcriptome measured by 5'-bromouridine immunoprecipitation chase sequencing (BRIC-seq), which enabled us to determine the half-lives of whole transcripts including lincRNAs, and we integrated BRIC-seq with ChIP-seq to achieve better estimation of the eventual transcript levels and to understand the importance of post-transcriptional regulation that determine the eventual transcript levels.
4.Hypobromous acid, a powerful endogenous electrophile: Experimental and theoretical studies.
Ximenes VF1, Morgon NH2, de Souza AR3. J Inorg Biochem. 2015 May;146:61-8. doi: 10.1016/j.jinorgbio.2015.02.014. Epub 2015 Mar 2.
Hypobromous acid (HOBr) is an inorganic acid produced by the oxidation of the bromide anion (Br(-)). The blood plasma level of Br(-) is more than 1,000-fold lower than that of chloride anion (Cl(-)). Consequently, the endogenous production of HOBr is also lower compared to hypochlorous acid (HOCl). Nevertheless, there is much evidence of the deleterious effects of HOBr. From these data, we hypothesized that the reactivity of HOBr could be better associated with its electrophilic strength. Our hypothesis was confirmed, since HOBr was significantly more reactive than HOCl when the oxidability of the studied compounds was not relevant. For instance: anisole (HOBr, k2=2.3×10(2)M(-1)s(-1), HOCl non-reactive); dansylglycine (HOBr, k2=7.3×10(6)M(-1)s(-1), HOCl, 5.2×10(2)M(-1)s(-1)); salicylic acid (HOBr, k2=4.0×10(4)M(-1)s(-1), non-reactive); 3-hydroxybenzoic acid (HOBr, k2=5.9×10(4)M(-1)s(-1), HOCl, k2=1.1×10(1)M(-1)s(-1)); uridine (HOBr, k2=1.