Abstract
The northwestern assay is employed to study the interaction between protein and RNA. The RNA binding proteins tend to bind to different kinds of RNA through either known domains or unknown sequences of proteins. Rice LGD1 recombinant protein, a grass-specific novel protein with RNA binding sequences in its C-terminal, was used to probe its function as an RNA binding protein. The LGD1 comprising von Willebrand factor type-A domain (vWA), coiled-coil and nuclear localization signal (NLS) is a class of protein that localizes both in the nucleus and cytoplasm. Although LGD1 does not contain any putative RNA binding domains, we could find high-affinity RNA binding residues at the C-terminus using ‘RNABindR’ prediction software (Terribilini et al., 2007). The LGD1 recombinant protein, purified from bacteria, somehow forms both dimer and monomer even under denaturing conditions. However, only the dimeric form is able to bind to total and mRNAs. Due to its reproducibility and reliability, we believe that this protocol can be used across different organisms.
Keywords: LGD1, Multiple transcripts, RNA binding, Alternative splicing, Rice
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Northwestern blot analysis of LGD1 recombinant protein. The recombinant GST-LGD1 was purified from E. coli and analyzed through SDS-PAGE, western and northwestern blots. (a) Coomassie Brilliant Blue (CBB) staining shows two major bands (arrows). The size of the lower band is ~56 kD (monomer) and upper band is ~112 kD (homodimer). (b) Anti-GST antibody recognizes the GST-LGD1 fusion protein (arrows) as well as GST (open circle). Northwestern blot shows the total RNA. (c) and mRNA (d) isolated from young panicle tissues bind to upper band (arrows), but not to the lower band (open arrowhead). Asterisks indicate RNA binding to degraded form of LGD1.
Recipes
Acknowledgments
We greatly appreciate the contributions of Drs Yue-Ie Hsing, Chyr-Guan Chern, Ming-Jen Fan and Su-May Yu to generate the TRIM database. We also thank Drs Ko Shimamoto (Nara Institute of Science and Technology, Japan), for sharing the pANDA-RNAi vector, and Su-May Yu (Institute of Molecular Biology, Academia Sinica), for excellent support with rice transgenic experiments. We also thank Ms Lin-Yun Kuang (Transgenic Plant Laboratory, Academia Sinica) for assistance in particle bombardment. We are grateful to Ms. Krisa Fredrickson for her English editing. This work was supported by research grants from Academia Sinica (Taiwan), the National Science and Technology Program for Agricultural Biotechnology (NSTP/AB, 098S0030055-AA, Taiwan), the National Science Council (98-2313-B-001-001-MY3, Taiwan) and the Li Foundation (USA) to Guang-Yuh, Jauh.
References
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