PP7-based mRNP purification was performed as previously described (Hogg and Goff 2010 (link)). For SDS-PAGE, complexes were eluted with RNase A/T1 Cocktail (AM2286; Ambion), diluted 1:50 in HLB150, as previously described (Hogg and Goff 2010 (link)). For mass spectrometry, samples were eluted in HLB supplemented with 1 M NaCl. For isolation of protein-stripped mRNAs, PP7-based mRNP purifications were performed and mRNA phenol-extracted using TRIzol (15596026; Invitrogen). Capped and polyadenylated in vitro transcribed NLuc RNA was prepared using ApaI-linearized NLuc template DNA and the mMessage mMachine T7 Transcription (AM1344; Ambion) and Poly(A) Tailing (AM1350; Ambion) kits according to manufacturer instructions. NLucβwtβTP and NLucβwtSMG5TP IVT mRNAs were prepared using PCR-amplified cDNA as a template for the in vitro transcription reactions (PCR primers: 5′ Forward: TAA TAC GAC TCA CTA TAG GGA GAC; 3′ Reverse: AGG AAA GGA CAG TGG GAG TG).
Protocol detail
Purification and Analysis of mRNPs
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Apai
Cdna
Isolation
Mass spectrometry
Mrnas
Mrnp
Nacl
Phenol
Primers
Protein
Rnase t1
Sds page
Transcription
Trizol
Corresponding Organization :
Other organizations : National Heart Lung and Blood Institute, National Institutes of Health
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Variable analysis
independent variables
- PP7-based mRNP purification method
- SDS-PAGE elution method (RNase A/T1 Cocktail dilution)
- Mass spectrometry elution method (HLB supplemented with 1 M NaCl)
- In vitro transcription of NLuc, NLucβwtβTP, and NLucβwtSMG5TP mRNAs
- Protein composition of purified mRNPs (analyzed by SDS-PAGE and mass spectrometry)
- Isolation of protein-stripped mRNAs
- Previously described protocols for PP7-based mRNP purification and SDS-PAGE elution (Hogg and Goff 2010)
- Use of Trizol for mRNA phenol extraction
- Use of ApaI-linearized NLuc template DNA and commercial kits (mMessage mMachine T7 Transcription and Poly(A) Tailing) for in vitro transcription
- Positive control: Capped and polyadenylated in vitro transcribed NLuc RNA
- Negative control: Not explicitly mentioned
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