Quantification of desired size RNA footprints in RIPiT elution. RNase I concentrations used are indicated at top of each lane nucleotide (nt) lengths are to the left. An autoradiogram of 26% denaturing PAGE with 5′ -labeled RNA fragments from base-hydrolysis of poly U 30 oligonucleotide (lane 1) or FLAG-Magoh:eIF4AIII RIPiT (lanes 2–5). Size distribution of EJC footprints upon RNase I titration. Proteins detected by western blot are indicated to the right. The stably expressed FLAG-tag fusion protein used in each sample is indicated directly above lane. The table on the top indicates the different fractions from the RIPiT procedure and the antibodies used for 1 st and 2 nd IPs. Levels of proteins detected by western blots in different fractions during EJC RIPiT.
The Tet concentration used for induction is indicated at the top of each lane protein identities are indicated to the right.
Western blots showing tetracycline (Tet)-mediated induction of eIF4AIII protein with the FLAG tag at its N- or C-terminus (top and bottom panels, respectively). Right: On the contrary, while CLIP reveals no information regarding the complexes an RNA-bound RBP is part of (crossed-out schematics), it can unveil the sites of direct contact between an RBP and RNA (bottom schematic).Ī.
However, RIPiT does not conclusively define direct RBP-RNA interactions (crossed-out schematic). Left: RIPiT can reveal the binding sites of an intact multi-subunit RNP, and can also distinguish between footprints of two compositionally similar complexes (schematics on gray background). RBPs (blue), non-RBPs (green) and proteins unique to each complex are shown (complex A: yellow complex B: red). Top: Two similar yet compositionally distinct hypothetical multi-subunit RNPs. RIPiT and CLIP yield different types of information. Levels of proteins detected by western blots in input (lanes 1–5) or in oligo-dT pulldown fractions from cells irradiated with the dosage of short-wave UV light indicated above each lane (lanes 6–10). Comparison of UV-crosslinkability of RBPs to polyA+ RNA. Note that the RNA bases are pointing away from the bound protein. Enlarged view of the RNA:protein interface. Crystal structure of eIF4AIII (PDB ID: 2J0S blue) complexed with RNA (red) and AMP-PNP. Aromatic residues (Phe-17 and Phe-59 from RRM1) that stack with the nucleobases are shown in cyan. Enlarged view of the DNA:protein interface. Crystal structure of two-RRM-containing UP1 domain of hnRNP A1 (PDB ID: 2UP1 blue) in complex with a target containing its AGGG preferred recognition motif (in this case, within single-stranded DNA, ssDNA red). It is therefore particularly suited for studying dynamic RNP assemblages whose composition evolves as gene expression proceeds.įormaldehyde crosslinking High-throughput sequencing Immunoprecipitation RIP-Seq RIPiT RNA binding proteins.Ĭopyright © 2013 Elsevier Inc. Further, among current high-throughput approaches, RIPiT has the unique capacity to differentiate binding sites of RNPs with overlapping protein composition. RIPiT-Seq is broadly applicable to all RBPs regardless of their RNA binding mode and thus provides a means to map the RNA binding sites of RBPs with poor inherent ultraviolet (UV) crosslinkability. RNA:protein immunoprecipitation in tandem (RIPiT) yields highly specific RNA footprints of cellular RNPs isolated via two sequential purifications the resulting RNA footprints can then be identified by high-throughput sequencing (Seq). Here we describe a ribonucleoprotein (RNP) footprinting approach we recently developed for identifying occupancy sites of both individual RBPs and multi-subunit RNP complexes. Development of high-throughput approaches to map the RNA interaction sites of individual RNA binding proteins (RBPs) transcriptome-wide is rapidly transforming our understanding of post-transcriptional gene regulatory mechanisms.
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