Splicing-independent recruitment of U1 snRNP to a transcription unit in living cells.
Numerous non-coding RNAs are known to be involved in the regulation of gene expression. In this work, we analyzed RNAs that co-immunoprecipitated with human RNA polymerase II from mitotic cell extracts and identified U1 small nuclear RNA (snRNA) as a major species. To investigate a possible splicing-independent recruitment of U1 snRNA to transcription units, we established cell lines having integrated a reporter gene containing a functional intron or a splicing-deficient construction. Recruitment of U snRNAs and some splicing factors to transcription sites was evaluated using fluorescence in situ hybridization (FISH) and immunofluorescence. To analyze imaging data, we developed a quantitative procedure, 'radial analysis', based on averaging data from multiple fluorescence images. The major splicing snRNAs (U2, U4 and U6 snRNAs) as well as the U2AF65 and SC35 splicing factors were found to be recruited only to transcription units containing a functional intron. By contrast, U1 snRNA, the U1-70K (also known as snRNP70) U1-associated protein as well as the ASF/SF2 (also known as SFRS1) serine/arginine-rich (SR) protein were efficiently recruited both to normally spliced and splicing-deficient transcription units. The constitutive association of U1 small nuclear ribonucleoprotein (snRNP) with the transcription machinery might play a role in coupling transcription with pre-mRNA maturation.
Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, Paris, France.
Béatrice
B
Spiluttini
Bo
B
Gu
Praveen
P
Belagal
Anna S
AS
Smirnova
Van Trung
VT
Nguyen
Charles
C
Hébert
Ute
U
Schmidt
Edouard
E
Bertrand
Xavier
X
Darzacq
Olivier
O
Bensaude
D002460:Cell Line; D002477:Cells / Q000166:cytology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D036641:Microscopy, Fluorescence, Multiphoton; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000378:metabolism; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D014158:Transcription, Genetic
D009687:Nuclear Proteins; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C490688:SNRNP70 protein, human; C405483:U1 small nuclear RNA; D000068103:Serine-Arginine Splicing Factors; D012319:RNA Polymerase II