The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing.
RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3' processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end.
The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel.
Yehuda
Y
Brody
Noa
N
Neufeld
Nicole
N
Bieberstein
Sebastien Z
SZ
Causse
Eva-Maria
EM
Böhnlein
Karla M
KM
Neugebauer
Xavier
X
Darzacq
Yaron
Y
Shav-Tal
D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007438:Introns; D055029:Inverted Repeat Sequences; D056968:Lac Repressors / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D055544:beta-Globins / Q000235:genetics
D056968:Lac Repressors; D012322:RNA Precursors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D017411:Ribonucleoproteins, Small Nuclear; D055544:beta-Globins; D049452:Green Fluorescent Proteins; D012319:RNA Polymerase II
Methods Enzymol. 2000;318:493-506
10890008
Nature. 2002 Apr 4;416(6880):499-506
11932736
FEBS Lett. 2002 Nov 6;531(2):109-14
12417296
Cell. 1979 Jul;17(3):725-35
225043
Cell. 2009 Feb 20;136(4):688-700
19239889
Mol Cell. 1998 Oct;2(4):437-45
9809065
Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3386-91
18287003
Mol Cell. 1999 Jun;3(6):697-705
10394358
J Cell Sci. 2010 May 15;123(Pt 10):1761-74
20427315
Science. 2004 Jun 18;304(5678):1797-800
15205532
Nat Cell Biol. 2010 Jun;12(6):543-52
20453848
J Cell Sci. 2002 Oct 15;115(Pt 20):3865-71
12244124
Nature. 2000 Apr 6;404(6778):604-9
10766243
Trends Biochem Sci. 2003 Apr;28(4):215-20
12713906
Nat Struct Mol Biol. 2008 Jan;15(1):71-8
18157150
Proc Natl Acad Sci U S A. 1999 Dec 21;96(26):14937-42
10611316
Curr Opin Cell Biol. 2005 Jun;17(3):332-9
15901505
EMBO J. 2004 Apr 21;23(8):1782-91
15057275
EMBO J. 2006 Aug 9;25(15):3469-79
16900099
Genes Dev. 2002 Nov 1;16(21):2792-9
12414732
Nature. 1997 May 29;387(6632):523-7
9168118
Mol Cell. 2006 Dec 28;24(6):903-15
17189192
Mol Cell. 2009 Oct 23;36(2):178-91
19854129
Proc Natl Acad Sci U S A. 2009 Oct 27;106(43):18357-61
19826084
Nat Struct Mol Biol. 2008 Aug;15(8):819-26
18641664
Genes Dev. 1991 Dec;5(12A):2288-302
1748285
Curr Opin Cell Biol. 2008 Jun;20(3):260-5
18436438
Nat Struct Mol Biol. 2009 Nov;16(11):1128-33
19820712
Nat Genet. 1995 Feb;9(2):184-90
7719347
Mol Cancer Ther. 2009 Aug;8(8):2308-18
19671752
Mol Cell. 2005 Jul 1;19(1):65-75
15989965
Nucleic Acids Res. 1996 Dec 1;24(23):4649-52
8972849
Mol Cell Biol. 2002 Oct;22(19):6706-18
12215528
Cell. 2004 Mar 5;116(5):683-98
15006351
Genes Dev. 1997 May 1;11(9):1148-59
9159396
Nucleic Acids Res. 1982 Sep 25;10(18):5429-45
6292841
J Cell Sci. 2010 Jun 15;123(Pt 12):2085-93
20519584
Trends Cell Biol. 2009 Aug;19(8):375-84
19616950
Trends Biochem Sci. 2010 Sep;35(9):497-504
20418102
Nature. 1994 Dec 22-29;372(6508):809-12
7997273
J Cell Biol. 2006 Apr 24;173(2):207-18
16618814
Genome Res. 2006 Jul;16(7):912-21
16769980
Nat Cell Biol. 2000 Dec;2(12):871-8
11146650
J Cell Biol. 2007 Oct 22;179(2):291-304
17954611
Mol Cell. 2008 Feb 1;29(2):271-8
18243121
FEBS Lett. 2008 Jun 18;582(14):1971-6
18435923
Nat Struct Mol Biol. 2007 Sep;14(9):796-806
17676063
J Cell Biol. 1999 Jun 28;145(7):1341-54
10385516
Nat Chem Biol. 2007 Sep;3(9):576-83
17643111
Nat Struct Biol. 2002 Nov;9(11):800-5
12389039
Mol Cell. 2003 Aug;12(2):525-32
14536091
RNA. 2002 Sep;8(9):1102-11
12358429
Nat Struct Mol Biol. 2006 Sep;13(9):815-22
16921380
Nat Struct Mol Biol. 2007 Sep;14(9):788-90
17823607
Nature. 2001 Feb 15;409(6822):860-921
11237011
Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7724-8
7644484
Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8253-7
8710856
Science. 2000 May 26;288(5470):1385-9
10827942
Mol Cell. 2006 Mar 17;21(6):849-59
16543153
Curr Opin Cell Biol. 2005 Jun;17(3):251-6
15901493
Mol Cell. 2007 Jun 22;26(6):867-81
17588520
Nature. 2001 Dec 20-27;414(6866):929-33
11780068
Genes Dev. 1988 Jun;2(6):754-65
3138163
Nucleic Acids Res. 2002 Jan 1;30(1):191-4
11752290
RNA. 2004 Oct;10(10):1489-98
15383674
J Cell Biol. 1997 Jan 13;136(1):19-28
9008700
Mol Cell. 2009 Apr 24;34(2):179-90
19394295
Mol Cell Biol. 1993 Oct;13(10):6211-22
8413221