RNA recognition motif 2 directs the recruitment of SF2/ASF to nuclear stress bodies.
Heat shock induces the transcriptional activation of large heterochromatic regions of the human genome composed of arrays of satellite III DNA repeats. A number of RNA-processing factors, among them splicing factor SF2/ASF, associate with these transcription factors giving rise to nuclear stress bodies (nSBs). Here, we show that the recruitment of SF2/ASF to these structures is mediated by its second RNA recognition motif. Amino acid substitutions in the first alpha-helix of this domain, but not in the beta-strand regions, abrogate the association with nSBs. The same mutations drastically affect the in vivo activity of SF2/ASF in the alternative splicing of adenoviral E1A transcripts. Sequence analysis identifies four putative high-affinity binding sites for SF2/ASF in the transcribed strand of the satellite III DNA. We have verified by gel mobility shift assays that the second RNA-binding domain of SF2/ASF binds at least one of these sites. Our analysis suggests that the recruitment of SF2/ASF to nSBs is mediated by a direct interaction with satellite III transcripts and points to the second RNA-binding domain of the protein as the major determinant of this interaction.
Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Via Abbiategrasso 207, 27100 Pavia, Italy.
Ilaria
I
Chiodi
Margherita
M
Corioni
Manuela
M
Giordano
Rut
R
Valgardsdottir
Claudia
C
Ghigna
Fabio
F
Cobianchi
Rui-Ming
RM
Xu
Silvano
S
Riva
Giuseppe
G
Biamonti
D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D022003:Cell Nucleus Structures / Q000378:metabolism*; D004276:DNA, Satellite / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D018869:Heat-Shock Response; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D021381:Protein Transport; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D020798:Two-Hybrid System Techniques
D004276:DNA, Satellite; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors