Optimized RNA gel-shift and UV cross-linking assays for characterization of cytoplasmic RNA-protein interactions

被引:46
作者
Thomson, AM
Rogers, JT
Walker, CE
Staton, JM
Leedman, PJ
机构
[1] Royal Perth Hosp, Lab Canc Med, Perth, WA 6001, Australia
[2] Brigham & Womens Hosp, Boston, MA 02115 USA
[3] Harvard Univ, Sch Med, Boston, MA USA
关键词
D O I
10.2144/99275rr03
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Considerable interest has recently focused on defining the mechanisms involved in the regulation of gene expression at the level of mRNA stability and translational efficiency. However the assays used to directly investigate interactions between RNA and cytoplasmic proteins have been difficult to establish, and methods are nor widely available. Here, we describe a robust method for RNA electrophoretic mobility shift and UV cross-linking assays that allows rapid detection of cytoplasmic RNA-protein interactions. For added convenience to new investigators, these assays use mini-gels with an electrophoresis time of 15-20 min, enabling a high throughput of samples. The method works successfully with many different probes and cytoplasmic extracts from a variety of cell lines. Furthermore, we provide a system to optimize characterization of the RNA-protein complex and troubleshoot most assay difficulties.
引用
收藏
页码:1032 / +
页数:9
相关论文
共 22 条
  • [1] Belasco J.G., 1993, Control of Messenger RNA Stability, P475
  • [2] EISENSTEIN RS, 1993, J BIOL CHEM, V268, P27363
  • [3] ENRIGHT C, 1994, RNA PROCESSING PRACT, V2, P155
  • [4] GILLIS P, 1991, J BIOL CHEM, V266, P3172
  • [5] Gu W, 1996, MOL CELL BIOL, V16, P4535
  • [6] HIGGINS SJ, 1994, RNA PROCESSING PRACT, V2
  • [7] Higgins SJ, 1994, RNA PROCESSING PRACT, VI
  • [8] p21waf1 mRNA contains a conserved element in its 3′-untranslated region that is bound by the Elav-like mRNA-stabilizing proteins
    Joseph, B
    Orlian, M
    Furneaux, H
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (32) : 20511 - 20516
  • [9] ROLE OF RNA SECONDARY STRUCTURE OF THE IRON-RESPONSIVE ELEMENT IN TRANSLATIONAL REGULATION OF FERRITIN SYNTHESIS
    KIKINIS, Z
    EISENSTEIN, RS
    BETTANY, AJE
    MUNRO, HN
    [J]. NUCLEIC ACIDS RESEARCH, 1995, 23 (20) : 4190 - 4195
  • [10] REGULATING THE FATE OF MESSENGER-RNA - THE CONTROL OF CELLULAR IRON-METABOLISM
    KLAUSNER, RD
    ROUAULT, TA
    HARFORD, JB
    [J]. CELL, 1993, 72 (01) : 19 - 28