The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

被引：81

作者：

Huranova, Martina ^{[1
]}

Ivani, Ivan ^{[1
]}

Benda, Ales ^{[2
]}

Poser, Ina ^{[3
]}

Brody, Yehuda ^{[4
,5
]}

Hof, Martin ^{[2
]}

Shav-Tal, Yaron ^{[4
,5
]}

Neugebauer, Karla M. ^{[3
]}

Stanek, David ^{[1
]}

机构：

[1] Acad Sci Czech Republic, Inst Mol Genet, CR-14220 Prague, Czech Republic

[2] Acad Sci Czech Republic, J Heyrovsky Inst Phys Chem, Prague 14220, Czech Republic

[3] Max Planck Inst Mol Cell Biol & Genet, D-01307 Dresden, Germany

[4] Bar Ilan Univ, Mina & Everard Goodman Fac Life Sci, IL-52900 Ramat Gan, Israel

[5] Bar Ilan Univ, Inst Nanotechnol & Adv Mat, IL-52900 Ramat Gan, Israel

来源：

JOURNAL OF CELL BIOLOGY | 2010年 / 191卷 / 01期

关键词：

FLUORESCENCE CORRELATION SPECTROSCOPY; IN-SITU TRANSCRIPTION; ASSEMBLY DYNAMICS; MAMMALIAN-CELLS; A-COMPLEXES; PENTA-SNRNP; U1; SNRNP; VIVO; SPLICEOSOME; BINDING;

D O I：

10.1083/jcb.201004030

中图分类号：

Q2 [细胞生物学];

学科分类号：

071009 ; 090102 ;

摘要：

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.

引用

页码：75 / 86

页数：12

共 63 条

[1] Analyses of In Vivo Interaction and Mobility of Two Spliceosomal Proteins Using FRAP and BiFC
Ali, Gul Shad
Prasad, K. V. S. K.
Hanumappa, M.
Reddy, A. S. N.
[J]. PLOS ONE, 2008, 3 (04):
[2] In vivo kinetics of mRNA splicing and transport in mammalian cells
Audibert, A
Weil, D
Dautry, F
[J]. MOLECULAR AND CELLULAR BIOLOGY, 2002, 22 (19) : 6706 - 6718
[3] Native spliceosomes assemble with Pre-mRNA to form supraspliceosomes
Azubel, M
Habib, N
Sperling, R
Sperling, J
[J]. JOURNAL OF MOLECULAR BIOLOGY, 2006, 356 (04) : 955 - 966
[4] Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes
Behzadnia, Nastaran
Golas, Monika M.
Hartmuth, Klaus
Sander, Bjoern
Kastner, Berthold
Deckert, Jochen
Dube, Prakash
Will, Cindy L.
Urlaub, Henning
Stark, Holger
Luerhrmann, Reinhard
[J]. EMBO JOURNAL, 2007, 26 (06) : 1737 - 1748
[5] Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP
Behzadnia, Nastaran
Hartmuth, Klaus
Will, Cindy L.
Luehrmann, Reinhard
[J]. RNA, 2006, 12 (09) : 1738 - 1746
[6] How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy
Benda, A
Benes, M
Marecek, V
Lhotsky, A
Hermens, WT
Hof, M
[J]. LANGMUIR, 2003, 19 (10) : 4120 - 4126
[7] SPLICE SITE SELECTION, RATE OF SPLICING, AND ALTERNATIVE SPLICING ON NASCENT TRANSCRIPTS
BEYER, AL
OSHEIM, YN
[J]. GENES & DEVELOPMENT, 1988, 2 (06) : 754 - 765
[8] AN ORDERED PATHWAY OF SNRNP BINDING DURING MAMMALIAN PRE-MESSENGER-RNA SPLICING COMPLEX ASSEMBLY
BINDEREIF, A
GREEN, MR
[J]. EMBO JOURNAL, 1987, 6 (08) : 2415 - 2424
[9] THE SPLICEOSOME - YEAST PRE-MESSENGER RNA ASSOCIATES WITH A 40S COMPLEX IN A SPLICING-DEPENDENT REACTION
BRODY, E
ABELSON, J
[J]. SCIENCE, 1985, 228 (4702) : 963 - 967
[10] A serine/arginine-rich domain in the human U1 70k protein is necessary and sufficient for ASF/SF2 binding
Cao, WH
Garcia-Blanco, MA
[J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (32) : 20629 - 20635

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