A detailed understanding of post-transcriptional gene expression is necessary to correlate the different elements involved in the many levels of RNA-protein interactions that are needed to coordinate the cellular biomolecular machinery. The profile of mRNA, a major component of this machinery, can be examined after isolation from specific RNA-binding proteins (RBPs). RIP-Chip or ribonomic profiling is a versatilein vivo technique that has been widely used to study post-transcriptional gene regulation and the localization of mRNA. Here we elaborately detail the methodology for mRNA isolation using RBP immunoprecipitation (RIP) as a primary approach. Specific antibodies are used to target RBPs, which are then used to capture the associated mRNA.
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"Small RNAs and their targets bind the Ago-containing RISC complexes, in which the Ago proteins form stable Ago ribonucleoproteins that can be biochemically analyzed [53,57,58]. The Ago-protein-binding small RNAs can be isolated by RIP [59,60]. In a previous work, BmAgo2 was fused with a HIS tag and was successfully expressed using the Baculovirus Bacmid system harboring the ie1 promoter enhanced with a hr5 enhancer . "
[Show abstract][Hide abstract] ABSTRACT: Small non-coding RNAs (ncRNAs) are important regulators of gene expression in eukaryotes. Previously, only microRNAs (miRNAs) and piRNAs have been identified in the silkworm, Bombyx mori. Furthermore, only ncRNAs (50-500nt) of intermediate size have been systematically identified in the silkworm.
Here, we performed a systematic identification and analysis of small RNAs (18-50nt) associated with the Bombyx mori argonaute2 (BmAgo2) protein. Using RIP-seq, we identified various types of small ncRNAs associated with BmAGO2. These ncRNAs showed a multimodal length distribution, with three peaks at ~20nt, ~27nt and ~33nt, which included tRNA-, transposable element (TE)-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. The tRNA-derived fragments (tRFs) were found at an extremely high abundance and accounted for 69.90% of the BmAgo2-associated small RNAs. Northern blotting confirmed that many tRFs were expressed or up-regulated only in the BmNPV-infected cells, implying that the tRFs play a prominent role by binding to BmAgo2 during BmNPV infection. Additional evidence suggested that there are potential cleavage sites on the D, anti-codon and TpsiC loops of the tRNAs. TE-derived small RNAs and piRNAs also accounted for a significant proportion of the BmAgo2-associated small RNAs, suggesting that BmAgo2 could be involved in the maintenance of genome stability by suppressing the activities of transposons guided by these small RNAs. Finally, Northern blotting was also used to confirm the Bombyx 5.8 s rRNA-derived small RNAs, demonstrating that various novel small RNAs exist in the silkworm.
Using an RIP-seq method in combination with Northern blotting, we identified various types of small RNAs associated with the BmAgo2 protein, including tRNA-, TE-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. Our findings provide new clues for future functional studies of the role of small RNAs in insect development and evolution.
"ENCODE RIP-Seq data analysis revealed that polyA-binding protein 1, Pabpc1 (Figure 4C), could be another candidate that preferentially associates with polyadenylation sites of short-isoforms. To ensure that the observed preference is not simply due to the mRNA binding ability of Pabpc1, the Pabpc1 profile was compared with that of a related mRNA binding protein Elav1 (58), performed using identical conditions. The comparisons reveal that the Pabpc1 is more preferentially enriched at short isoform locations, suggesting that Pabpc1 likely has a role in regulating the polyadenylation sites of short isoforms (Figure 4C). "
[Show abstract][Hide abstract] ABSTRACT: We present a comprehensive map of over 1 million polyadenylation sites and quantify their usage in major cancers and tumor cell lines using direct RNA sequencing. We built the Expression and Polyadenylation Database to enable the visualization of the polyadenylation maps in various cancers and to facilitate the discovery of novel genes and gene isoforms that are potentially important to tumorigenesis. Analyses of polyadenylation sites indicate that a large fraction (∼30%) of mRNAs contain alternative polyadenylation sites in their 3' untranslated regions, independent of the cell type. The shortest 3' untranslated region isoforms are preferentially upregulated in cancer tissues, genome-wide. Candidate targets of alternative polyadenylation-mediated upregulation of short isoforms include POLR2K, and signaling cascades of cell-cell and cell-extracellular matrix contact, particularly involving regulators of Rho GTPases. Polyadenylation maps also helped to improve 3' untranslated region annotations and identify candidate regulatory marks such as sequence motifs, H3K36Me3 and Pabpc1 that are isoform dependent and occur in a position-specific manner. In summary, these results highlight the need to go beyond monitoring only the cumulative transcript levels for a gene, to separately analysing the expression of its RNA isoforms.
Nucleic Acids Research 06/2012; 40(17):8460-71. DOI:10.1093/nar/gks637 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Classical zinc fingers (ZFs) are one of the most common protein domains in higher eukaryotes and have been known for almost 30 years to act as sequence-specific DNA-binding domains. This knowledge has come, however, from the study of a small number of archetypal proteins, and a larger picture is beginning to emerge that ZF functions are far more diverse than originally suspected. Here, we review the evidence that a subset of ZF proteins live double lives, binding to both DNA and RNA targets and frequenting both the cytoplasm and the nucleus. This duality can create an important additional level of gene regulation that serves to connect transcriptional and post-transcriptional control.