PMID- 35965005
OWN - NLM
STAT- MEDLINE
DCOM- 20220816
LR  - 20220823
IS  - 1557-7988 (Electronic)
IS  - 0076-6879 (Linking)
VI  - 673
DP  - 2022
TI  - Lysate and cell-based assays to probe the translational role of RNA helicases.
PG  - 141-168
LID - S0076-6879(22)00123-9 [pii]
LID - 10.1016/bs.mie.2022.03.032 [doi]
AB  - Translation initiation is the first step in protein synthesis, during which the 
      small subunit of the ribosome scans the 5' untranslated region (5'UTR) of an mRNA 
      to identify a start codon and commence translation elongation. By unwinding and 
      modulating secondary structures and other RNA features present in the 5'UTR, RNA 
      helicases can regulate ribosome scanning and start codon selection. This chapter 
      presents an approach to measure the effect of RNA helicases on mRNA translation 
      initiation. 5'UTR luciferase reporters are transcribed in vitro and employed in 
      either of two assays. The in vitro assay translates the reporters in a cell-free 
      whole-cell lysate system, which allows for greater biochemical manipulation and 
      tighter control over confounding effects. In the alternative cell-based approach, 
      the reporters are transfected and translated in living cells, which provides a 
      more physiological setup. Either method can be used to investigate how the 
      perturbation of a helicase, such as changes in protein levels or mutations, 
      affects translation initiation at the 5'UTR level. The chapter also discusses 
      alternative approaches, troubleshooting, and further applications of these 
      methods. These assays will provide insights on the role of helicases and other 
      translational factors as regulators of the proteome both in physiological and 
      diseased settings.
CI  - Copyright (c) 2022 Elsevier Inc. All rights reserved.
FAU - Wilkins, Kevin C
AU  - Wilkins KC
AD  - Department of Cell and Tissue Biology, University of California, San Francisco, 
      CA, United States; Graduate Division, University of California, San Francisco, 
      San Francisco, CA, United States.
FAU - Venkataramanan, Srivats
AU  - Venkataramanan S
AD  - Department of Cell and Tissue Biology, University of California, San Francisco, 
      CA, United States.
FAU - Floor, Stephen N
AU  - Floor SN
AD  - Department of Cell and Tissue Biology, University of California, San Francisco, 
      CA, United States; Helen Diller Family Comprehensive Cancer Center, University of 
      California, San Francisco, San Francisco, CA, United States. Electronic address: 
      stephen@floorlab.org.
LA  - eng
GR  - DP2 GM132932/GM/NIGMS NIH HHS/United States
GR  - R01 NS120667/NS/NINDS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20220419
PL  - United States
TA  - Methods Enzymol
JT  - Methods in enzymology
JID - 0212271
RN  - 0 (5' Untranslated Regions)
RN  - 0 (Codon, Initiator)
RN  - EC 3.6.4.13 (RNA Helicases)
SB  - IM
MH  - 5' Untranslated Regions
MH  - Codon, Initiator
MH  - Peptide Chain Initiation, Translational
MH  - *Protein Biosynthesis
MH  - *RNA Helicases/genetics
OTO - NOTNLM
OT  - DEAD-box proteins
OT  - Luciferase assay
OT  - Protein synthesis
OT  - RNA chaperones
OT  - Translational control
EDAT- 2022/08/15 06:00
MHDA- 2022/08/17 06:00
CRDT- 2022/08/14 21:04
PHST- 2022/08/14 21:04 [entrez]
PHST- 2022/08/15 06:00 [pubmed]
PHST- 2022/08/17 06:00 [medline]
AID - S0076-6879(22)00123-9 [pii]
AID - 10.1016/bs.mie.2022.03.032 [doi]
PST - ppublish
SO  - Methods Enzymol. 2022;673:141-168. doi: 10.1016/bs.mie.2022.03.032. Epub 2022 Apr 
      19.