Retinitis pigmentosa (RP) is a hereditary disorder caused by mutations in more than 70 different genes including those that encode proteins important for pre-mRNA splicing. Most RP-associated mutations in splicing factors reduce either their expression, stability or incorporation into functional splicing complexes. However, we have previously shown that two RP mutations in PRPF8 (F2314L and Y2334N) and two in SNRNP200 (S1087L and R1090L) behaved differently, and it was still unclear how these mutations affect the functions of both proteins. To investigate this in the context of functional spliceosomes, we used iCLIP in HeLa and retinal pigment epithelial (RPE) cells. We found that both mutations in the RNA helicase SNRNP200 change its interaction with U4 and U6 snRNAs. The significantly broader binding profile of mutated SNRNP200 within the U4 region upstream of the U4/U6 stem I strongly suggests that its activity to unwind snRNAs is impaired. This was confirmed by FRAP measurements and helicase activity assays comparing mutant and WT protein. The RP variants of PRPF8 did not affect snRNAs, but showed a reduced binding to pre-mRNAs, which resulted in the slower splicing of introns and altered expression of hundreds of genes in RPE cells. This suggests that changes in the expression and splicing of specific genes are the main driver of retinal degeneration in PRPF8-linked RP.

• MeSH
• HeLa Cells MeSH
• Humans MeSH
• Ribonucleoprotein, U4-U6 Small Nuclear metabolism   genetics   MeSH
• Mutation * MeSH
• Eye Proteins genetics   metabolism   MeSH
• RNA Precursors * metabolism   genetics   MeSH
• RNA-Binding Proteins metabolism   genetics   MeSH
• Retinal Pigment Epithelium metabolism   pathology   MeSH
• Retinitis Pigmentosa * genetics   metabolism   pathology   MeSH
• Ribonucleoproteins, Small Nuclear metabolism   genetics   MeSH
• RNA, Small Nuclear genetics   metabolism   MeSH
• RNA Splicing * genetics   MeSH
• Spliceosomes metabolism   genetics   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Replication-dependent histones (RDH) are required for packaging of newly synthetized DNA into nucleosomes during the S phase when their expression is highly upregulated. However, the mechanisms of this upregulation in metazoan cells remain poorly understood. Using iCLIP and ChIP-seq, we found that human cyclin-dependent kinase 11 (CDK11) associates with RNA and chromatin of RDH genes primarily in the S phase. Moreover, its amino-terminal region binds FLASH, an RDH-specific 3'-end processing factor, which keeps the kinase on the chromatin. CDK11 phosphorylates serine 2 (Ser2) of the carboxy-terminal domain of RNA polymerase II (RNAPII), which is initiated when RNAPII reaches the middle of RDH genes and is required for further RNAPII elongation and 3'-end processing. CDK11 depletion leads to decreased number of cells in S phase, likely owing to the function of CDK11 in RDH gene expression. Thus, the reliance of RDH expression on CDK11 could explain why CDK11 is essential for the growth of many cancers.

• MeSH
• Chromatin genetics   metabolism   MeSH
• Cyclin-Dependent Kinases genetics   metabolism   MeSH
• Phosphorylation MeSH
• Transcription, Genetic * MeSH
• Histones genetics   metabolism   MeSH
• Humans MeSH
• Apoptosis Regulatory Proteins genetics   metabolism   MeSH
• Calcium-Binding Proteins genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• DNA Replication MeSH
• RNA genetics   metabolism   MeSH
• S Phase MeSH
• Serine metabolism   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• 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

Many nascent long non-coding RNAs (lncRNAs) undergo the same maturation steps as pre-mRNAs of protein-coding genes (PCGs), but they are often poorly spliced. To identify the underlying mechanisms for this phenomenon, we searched for putative splicing inhibitory sequences using the ncRNA-a2 as a model. Genome-wide analyses of intergenic lncRNAs (lincRNAs) revealed that lincRNA splicing efficiency positively correlates with 5'ss strength while no such correlation was identified for PCGs. In addition, efficiently spliced lincRNAs have higher thymidine content in the polypyrimidine tract (PPT) compared to efficiently spliced PCGs. Using model lincRNAs, we provide experimental evidence that strengthening the 5'ss and increasing the T content in PPT significantly enhances lincRNA splicing. We further showed that lincRNA exons contain less putative binding sites for SR proteins. To map binding of SR proteins to lincRNAs, we performed iCLIP with SRSF2, SRSF5 and SRSF6 and analyzed eCLIP data for SRSF1, SRSF7 and SRSF9. All examined SR proteins bind lincRNA exons to a much lower extent than expression-matched PCGs. We propose that lincRNAs lack the cooperative interaction network that enhances splicing, which renders their splicing outcome more dependent on the optimality of splice sites.

• MeSH
• HeLa Cells MeSH
• Introns * MeSH
• Humans MeSH
• RNA Splice Sites * MeSH
• Pyrimidines analysis   MeSH
• RNA, Long Noncoding metabolism   MeSH
• Serine-Arginine Splicing Factors metabolism   MeSH
• RNA Splicing * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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.

• 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