RNA-binding proteins (RBPs) are critical to posttranscriptional gene regulation. Therefore, characterization of the RNA molecules bound by RBPs in vivo represent a key step in elucidating their function. The recently developed iCLIP technique allows single nucleotide resolution of the RNA binding footprints of RBPs. We present the iCLIP technique modified for its application to Trypanosoma brucei and most likely other kinetoplastid flagellates. By using the immuno- or affinity purification approach, it was successfully applied to the analysis of several RBPs. Furthermore, we also provide a detailed description of the iCLIP/iCLAP protocol that shall be particularly suitable for the studies of trypanosome RBPs.

• Keywords
• Posttranscriptional gene regulation, RNA-binding proteins (RBPs), iCLAP, iCLIP,
• MeSH
• Immunoprecipitation methods   MeSH
• Nucleotides genetics   metabolism   MeSH
• Parasitology methods   MeSH
• RNA-Binding Proteins analysis   genetics   metabolism   MeSH
• Protozoan Proteins analysis   genetics   metabolism   MeSH
• RNA, Protozoan genetics   metabolism   MeSH
• RNA genetics   metabolism   MeSH
• Trypanosoma brucei brucei genetics   MeSH
• Ultraviolet Rays MeSH
• Protein Binding genetics   radiation effects   MeSH
• Binding Sites genetics   MeSH
• Single Molecule Imaging methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Nucleotides MeSH
• RNA-Binding Proteins MeSH
• Protozoan Proteins MeSH
• RNA, Protozoan MeSH
• RNA MeSH

Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3' extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3' adenylation, uridine insertion/deletion editing, and 3' A/U-tailing. The rRNAs and gRNAs are 3' uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.

• Keywords
• RNA decay, RNA editing, Trypanosoma, kinetoplast, mitochondria, polyadenylation,
• MeSH
• RNA Editing physiology   MeSH
• RNA, Mitochondrial genetics   metabolism   MeSH
• RNA, Protozoan genetics   metabolism   MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• RNA, Mitochondrial MeSH
• RNA, Protozoan MeSH

MRP1/2 is a heteromeric protein complex that functions in the trypanosomatid mitochondrion as part of the RNA editing machinery, which facilitates multiple targeted insertions and deletions of uridines. MRP1/2 was shown to interact with MRB8170, which initiates RNA editing by marking pre-edited mRNAs, while TbRGG2 is required for its efficient progression on pan-edited mRNAs. Both MRP1/2 and TbRGG2 are capable of modulating RNA-RNA interactions in vitro. As determined by using iCLIP and RIP-qPCR, RNAs bound to MRP1/2 are characterized and compared with those associated with MRB8170 and TbRGG2. We provide evidence that MRP1 and MRB8170 have correlated binding and similar RNA crosslinking peak profiles over minimally and never-edited mRNAs. Our results suggest that MRP1 assists MRB8170 in RNA editing on minimally edited mRNAs.

• Keywords
• RNA binding proteins, RNA editing, iCLIP, mitochondrion, ribonuclear protein, trypanosome,
• MeSH
• RNA Editing MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mitochondria genetics   metabolism   MeSH
• RNA-Binding Proteins metabolism   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• RNA, Mitochondrial genetics   metabolism   MeSH
• Trypanosoma genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• gBP21 protein, Trypanosoma brucei MeSH Browser
• RNA, Messenger MeSH
• mitochondrial messenger RNA MeSH Browser
• RNA-Binding Proteins MeSH
• Protozoan Proteins MeSH
• RNA, Mitochondrial MeSH