Project description:Nucleotides on RNA are chemically modified during biogenesis of various mammalian RNA species and RNA modifications have profound effects on RNA function. The 2,2,7-trimethylguanosine modification of the 5′ of RNA molecules is one of the earliest discovered modified nucleotides on mammalian RNAs. Although the TMG cap is one of the most intensively studied RNA modifications, its distribution on a global scale has not been explored. In this study, the locations of the TMG cap on human RNA is determined, using RNA immunoprecipitation coupled with high-throughput sequencing. Although all prototypical modification sites were supported by this study, modification positions of some RNAs were erroneously annotated. In contrast to prototypic sites, many sites reported later were not validated at all. In addition to annotated modification sites, novel candidate sites were found on small Cajal body-specific RNAs.
Project description:A series of trinucleotide cap analogs functionalized with an amine-terminated linker at the guanosine ribose and immobilized on BrCN-activated Sepharose was prepared. These affinity resins AR-1 (Am), AR-2 (m6Am), and AR-3 (Bn6Am) were incubated with HEK293F cell extract in the presence of GTP to limit non-specific interactions. The pulled-down proteins were eluted with the corresponding trinucleotide cap analog (m7GpppAmpG for AR-1, m7Gpppm6AmpG for AR-2, and m7GpppBn6AmpG for AR-3), digested with trypsin, labeled with isobaric tags (TMT), and analyzed by shotgun proteomics. This experiment was performed in order to assess binding preferences of proteins to the resins AR-1, AR-2, and AR-3.
Project description:We find guanosine supplementation can induce metabolic stress on cells. To evaluate this effect we use RKO-NC and RKO-shPNP cells for experiment.
Project description:To annotate estrogen-regulated eRNAs in MCF-7 breast cancer cells, we used precision nuclear run-on and sequencing of capped RNA (PRO-cap) to determine the transcription start sites of eRNAs.
Project description:Two decades into the genomics era the question of mapping sequence to function has evolved from identifying functional elements to characterizing their quantitative properties including, in particular, their specificity and efficiency. Here, we use a large-scale approach to establish a quantitative map between the sequence of a bacterial regulatory RNA and its efficiency in modulating the expression of its targets. Our approach generalizes the sort-seq method, introduced recently to analyze promoter sequences, in order to accurately quantify the efficiency of a large library of sequence variants. We focus on two small RNAs (sRNAs) in E. coli, DsrA and RyhB, and their regulation of both repressed and activated targets. In addition to precisely identifying functional elements in the sRNAs, our data establish quantitative relationships between structural and energetic features of the sRNAs and their regulatory activity, and characterize a large set of direct and indirect interactions between nucleotides. A core of these interactions supports a model where specificity can be enhanced by a rigid molecular structure. Both sRNAs exhibit a modular design with limited cross-interactions, dividing the requirements for structural stability and target binding among modules.