Analyses of in vivo interaction and mobility of two spliceosomal proteins using FRAP and BiFC.

PloS One
Gul Shad AliA S N Reddy

Abstract

U1-70K, a U1 snRNP-specific protein, and serine/arginine-rich (SR) proteins are components of the spliceosome and play critical roles in both constitutive and alternative pre-mRNA splicing. However, the mobility properties of U1-70K, its in vivo interaction with SR proteins, and the mobility of the U1-70K-SR protein complex have not been studied in any system. Here, we studied the in vivo interaction of U1-70K with an SR protein (SR45) and the mobility of the U1-70K/SR protein complex using bimolecular fluorescence complementation (BiFC) and fluorescence recovery after photobleaching (FRAP). Our results show that U1-70K exchanges between speckles and the nucleoplasmic pool very rapidly and that this exchange is sensitive to ongoing transcription and phosphorylation. BiFC analyses showed that U1-70K and SR45 interacted primarily in speckles and that this interaction is mediated by the RS1 or RS2 domain of SR45. FRAP analyses showed considerably slower recovery of the SR45/U1-70K complex than either protein alone indicating that SR45/U1-70K complexes remain in the speckles for a longer duration. Furthermore, FRAP analyses with SR45/U1-70K complex in the presence of inhibitors of phosphorylation did not reveal any significant chan...Continue Reading

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Citations

Dec 23, 2008·PloS One·Zdravko J Lorković, Andrea Barta
Nov 3, 2011·Plant Physiology·Anireddy S N ReddyAndrea Barta
Sep 11, 2012·Protoplasma·Raquel F CarvalhoPaula Duque
Feb 25, 2009·Plant Molecular Biology·Noriaki TanabeShigeru Shigeoka
Oct 6, 2010·The Journal of Cell Biology·Martina HuranováDavid Stanek
May 9, 2012·The Plant Journal : for Cell and Molecular Biology·Irene S DayAnireddy S N Reddy
Nov 3, 2010·Mass Spectrometry Reviews·Ganesh Kumar AgrawalRandeep Rakwal
Jul 19, 2011·Wiley Interdisciplinary Reviews. RNA·Anireddy S N Reddy, Gul Shad Ali
Apr 13, 2013·Nucleus·Jonathan Sheinberger, Yaron Shav-Tal
Nov 26, 2009·Journal of Integrative Neuroscience·Peter A Bandettini
Aug 21, 2010·Plant Signaling & Behavior·Tianbao YangB W Poovaiah
Apr 11, 2019·Plant Molecular Biology·Mohammed AlbaqamiAnireddy S N Reddy

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Methods Mentioned

BETA
two
pull-down
fluorescence complementation
Fluorescence recovery after photobleaching
Fluorescence
transfection
PCR

Software Mentioned

GraphPad Prism
LSM

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