Spatial mapping of splicing factor complexes involved in exon and intron definition

J. D. Ellis, D. Llères, M. Denegri, A. Lamond, J. Cáceres

Research output: Contribution to journalArticle

Abstract

Nuclear pre-mRNA splicing occurs in eukaryotic cells in order to remove the intervening sequences (introns) that interrupt coding regions (exons) of genes. This pre-mRNA splicing occurs in the spliceosome (a quite large RNP complex), the assembly of which starts upon recognition of the 5′ and 3′ splice sites by the Ul small nuclear ribonucleoprotein (snRNP) and U2 snRNP auxiliary factor (U2AF), respectively. In addition to the small RNP particles Ul, U2, U4/U6, and U5 snRNPs, numerous non-snRNP splicing factors constitute the spliceosome, among which is the SR family of proteins, which have a modular domain structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal RS domain rich in arginine and serine residues. The RRMs determine RNA binding specificity, while the RS domain mediates protein-protein interactions. In this study, fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) techniques were used to determine protein-protein interaction between splicing factors in nanometer resolutions. The interaction of splicing factor 2/alternative splicing factor (SF2/ASF) with Ul 70K (the Ul snRNP-associated protein) in live HeLa cells was revealed via FRET acceptor photobleaching microscopy. After photobleaching, a strong FRET signal was detected in HeLa cells coexpressing ECFP-U1 70K and EYFP- SF2/ASF (Fig. 1), indicating that the two proteins can interact in vivo. Treatment with DRB, a transcription inhibitor, did not prevent Ul 70K and SF2/ ASF interaction in HeLa cells, demonstrating that their interaction does not occur exclusively upon pre-mRNA. SF2/ASF interaction with U2AF35 occurs as speckles in the nucleus of HeLa cells. The researchers have also shown that both SC35 and SRp20 interact with Ul 70K and U2AF35 in live HeLa cells. DRB treatment also did not prevent their interaction. A novel interaction of HCC1, a protein factor highly homologous to U2AF65, with U2AF35 and with U2AF65 was also revealed by Co-IP experiments and FRET microscopy.

Original languageEnglish
Pages (from-to)243-244
Number of pages2
JournalChemtracts
Volume21
Issue number6
Publication statusPublished - Jun 2008

Fingerprint

Introns
Exons
Fluorescence Resonance Energy Transfer
HeLa Cells
RNA Precursors
Alternative Splicing
Dichlororibofuranosylbenzimidazole
Photobleaching
Microscopic examination
RNA Splice Sites
Proteins
RNA
Small Nuclear Ribonucleoproteins
Spliceosomes
Microscopy
U2 Small Nuclear Ribonucleoproteins
Protein Interaction Domains and Motifs
Ribonucleoproteins
Transcription
Speckle

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry
  • Molecular Biology

Cite this

Ellis, J. D., Llères, D., Denegri, M., Lamond, A., & Cáceres, J. (2008). Spatial mapping of splicing factor complexes involved in exon and intron definition. Chemtracts, 21(6), 243-244.

Spatial mapping of splicing factor complexes involved in exon and intron definition. / Ellis, J. D.; Llères, D.; Denegri, M.; Lamond, A.; Cáceres, J.

In: Chemtracts, Vol. 21, No. 6, 06.2008, p. 243-244.

Research output: Contribution to journalArticle

Ellis, JD, Llères, D, Denegri, M, Lamond, A & Cáceres, J 2008, 'Spatial mapping of splicing factor complexes involved in exon and intron definition', Chemtracts, vol. 21, no. 6, pp. 243-244.
Ellis JD, Llères D, Denegri M, Lamond A, Cáceres J. Spatial mapping of splicing factor complexes involved in exon and intron definition. Chemtracts. 2008 Jun;21(6):243-244.
Ellis, J. D. ; Llères, D. ; Denegri, M. ; Lamond, A. ; Cáceres, J. / Spatial mapping of splicing factor complexes involved in exon and intron definition. In: Chemtracts. 2008 ; Vol. 21, No. 6. pp. 243-244.
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