Project description:Signaling pathways are controlled by a vast array of post-translational mechanisms. By contrast, little is known regarding the mechanisms that regulate the expression of their core components. We conducted an RNAi screen in Drosophila for factors modulating RAS/MAPK signaling and identified the Exon Junction Complex (EJC) as a novel key element of this pathway. The EJC binds the exon-exon junctions of mRNAs, and thus far, has been linked exclusively to post-splicing events. Here, we report that the EJC is required for proper splicing of mapk transcripts by a mechanism that apparently controls exon definition. Moreover, whole transcriptome and RT-PCR analyses of EJC-depleted cells revealed that the splicing of long intron-containing genes, which includes mapk, is sensitive to EJC activity. These results identify a role for the EJC in the splicing of a subset of transcripts and suggest that RAS/MAPK signaling depends on the regulation of MAPK levels by the EJC. Transcriptome sequencing (RNA-Seq) of Drosophila S2 cells to monitor the effect of EJC depletion on the cellular mRNA expression profile. Each treatment (dsRNA knockdown of MAGO (CG9401), dsRNA knockdown of eIF4AIII (CG7483)) was done in biological duplicate and each sample was sequenced separately on a quad slide on the SOLiD 3.0 platform. The reference samples were treated with a dsRNA targeted to GFP.
Project description:RNA was purified from serum of osteoporotic and healthy postmenopausal mexican women using miRNeasy Serum/Plasma kit (QIAGEN). For microRNA expression analysis we used the Human MicroRNA A+B Cards Set v3.0 TaqMan Low Density Array platform (Applied Biosystems). Analysis was performed in the Expression Suite v.1.1.3 software (Applied Biosystems).
Project description:Total RNA extracted from the head and neck cell line CAL165 were purified using miRNeasy minikit (Qiagen). RNA libraries were then generated with the NEB next small library prep set for SOLID (New England Biolabs) and sequenced on the Applied Biosystems SOLiD 5500 wildfire system following the manufacturer's instructions.
Project description:Total RNA extracted from biopsies of healthy skin, papilloma, SCC Tumor and of 2 cell lines isolated from these tumors were purified using miRNeasy minikit (Qiagen). RNA libraries were then generated with the NEB next small library prep set for SOLID (New England Biolabs) and sequenced on the Applied Biosystems SOLiD 5500 wildfire system following the manufacturer's instructions.
Project description:Twenty-four triple-negative breast cancer and 14 adjacent normal tissues were collected from breast cancer patients during surgeries at National Taiwan University Hospital (NTUH, Taipei, Taiwan). All triple-negative breast cancer samples were invasive ductal carcinomas (IDC) and were negative in immunohistochemical statuses of ER, PR, and HER2 receptors, as confirmed by professional pathologists. Treatment procedure of all patients followed the National Comprehensive Cancer Network (NCCN) guideline. All samples were neoadjuvant-free and were collected before systemic chemotherapy treatments. Written informed consent was obtained from all patients who participated in this study. Using human tissues for research in this study was approved by the institutional review board at NTUH. A novel set of 25-miRNA signature identified in this study was able to effectively distinguish between triple-negative breast cancer and adjacent normal tissues. Moreover, we documented the first evidence of seven polycistronic miRNA clusters preferentially harboring deregulated miRNA genes in triple-negative breast cancer. In the present study, a panel of 24 triple-negative breast cancer and 14 adjacent normal tissue samples were examined for the presence of deregulated miRNA genes using the high-throughput sequencing technology. Total RNA was extracted from the triple-negative breast cancer and adjacent normal samples for preparation of small RNA libraries. Each small RNA library was constructed from total RNA of each sample using the SOLiD Total RNA-Seq Kit (Applied Biosystems, Foster City, CA, USA). Upon completion of polymerase chain reaction (PCR) amplification, small RNA libraries were purified using the SOLiD Library Micro Column Purification Kit (Applied Biosystems) and hybridized to the template beads using the SOLiD EZ bead system (Applied Biosystems). The template beads were amplified and deposited onto subtract for ligation sequencing by SOLiD 4 System (Applied Biosystems).
Project description:Promoter-proximal pausing of RNA polymerase II (Pol II) is a widespread transcriptional regulatory step across metazoans. Here we find that the nuclear exon junction complex (pre-EJC) is a critical and conserved regulator of this process. Depletion of pre-EJC subunits leads to a global decrease in Pol II pausing and to premature entry into elongation. This effect occurs, at least in part, via non-canonical recruitment of pre-EJC components at promoters. Failure to recruit the pre-EJC at promoters results in increased binding of the positive transcription elongation complex (P-TEFb) and in enhanced Pol II release. Notably, restoring pausing is sufficient to rescue exon skipping and the photoreceptor differentiation defect associated with depletion of pre-EJC components in vivo. We propose that the pre-EJC serves as an early transcriptional checkpoint to prevent premature entry into elongation, ensuring proper recruitment of RNA processing components that are necessary for exon definition.