Synthesis and Labeling of RNA In Vitro
This unit discusses several methods for generating large amounts of uniformly labeled, end-labeled, and site-specifically labeled RNAs in vitro. The methods involve a number of experimental procedures, including RNA transcription, 5' dephosphorylation and rephosphorylation, 3' terminal nucleotide addition (via ligation), site-specific RNase H cleavage directed by 2'-O-methyl RNA-DNA chimeras, and 2-piece splint ligation. The applications of these RNA radiolabeling approaches are also discussed. Curr. Protoc. Mol. Biol. 102:4.15.1-4.15.14. © 2013 by John Wiley & Sons, Inc.
- [Show abstract] [Hide abstract] ABSTRACT: This unit discusses a basic method for purification of radiolabeled RNAs using denaturing polyacrylamide gel electrophoresis. The method consists of a number of experimental procedures, including total RNA preparation from yeast cells, isolation of a specific RNA from total yeast RNA, RNA 3'-terminal labeling using nucleotide (5' [(32) P]pCp) addition (via ligation), denaturing (8 M urea) polyacrylamide gel electrophoresis, and RNA extraction from the gel slice. Key points for achieving good electrophoretic separation of RNA are also discussed. Curr. Protoc. Mol. Biol. 105:4.20.1-4.20.13. © 2014 by John Wiley & Sons, Inc.0Comments 1Citation
- [Show abstract] [Hide abstract] ABSTRACT: Poly(ADP-ribose) polymerase-13 (PARP13/ZAP/ZC3HAV1) is an antiviral factor, active against specific RNA viruses such as murine leukaemia virus, Sindbis virus and human immunodeficiency virus. During infection, PARP13 binds viral RNA via its four CCCH-type zinc-finger domains and targets it for degradation by recruiting cellular messenger RNA (mRNA) decay factors such as the exosome complex and XRN1. Here we show that PARP13 binds to and regulates cellular mRNAs in the absence of viral infection. Knockdown of PARP13 results in the misregulation of hundreds of transcripts. Among the most upregulated transcripts is TRAILR4 that encodes a decoy receptor for TRAIL-a pro-apoptotic cytokine that is a promising target for the therapeutic inhibition of cancers. PARP13 destabilizes TRAILR4 mRNA post-transcriptionally in an exosome-dependent manner by binding to a region in its 3' untranslated region. As a consequence, PARP13 represses TRAILR4 expression and increases cell sensitivity to TRAIL-mediated apoptosis, acting as a key regulator of the cellular response to TRAIL.0Comments 8Citations
- [Show abstract] [Hide abstract] ABSTRACT: Centrally located at the ribosomal subunit interface and mRNA tunnel, helix 69 (H69) from 23S rRNA participates in key steps of translation. Ribosome activity is influenced by three pseudouridine modifications, which modulate the structure and conformational behavior of H69. To understand how H69 is affected by the presence of pseudouridine in combination with sequence changes, the biophysical properties of wild-type H69 and representative mutants (A1912G, U1917C, and A1919G) were examined. Results from NMR and circular dichroism spectroscopy indicate that pH-dependent structural changes of wild-type H69 and the chosen mutants are modulated by pseudouridine and loop sequence. The effects of the mutations on global stability of H69 are negligible; however, pseudouridine stabilizes H69 at low pH conditions. Alterations to induced conformational changes of H69 likely result in compromised function, as indicated by previous biological studies. Copyright © 2015 Elsevier B.V. All rights reserved.0Comments 0Citations