RNA looping by PTB: Evidence using FRET and NMR spectroscopy for a role in splicing repression.
Alternative splicing plays an important role in generating proteome diversity. The polypyrimidine tract-binding protein (PTB) is a key alternative splicing factor involved in exon repression. It has been proposed that PTB acts by looping out exons flanked by pyrimidine tracts. We present fluorescence, NMR, and in vivo splicing data in support of a role of PTB in inducing RNA loops. We show that the RNA recognition motifs (RRMs) 3 and 4 of PTB can bind two distant pyrimidine tracts and bring their 5' and 3' ends in close proximity, thus looping the RNA. Efficient looping requires an intervening sequence of 15 nucleotides or longer between the pyrimidine tracts. RRM3 and RRM4 bind the 5' and the 3' pyrimidine tracts, respectively, in a specific directionality and work synergistically for efficient splicing repression in vivo.
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.
Rajan
R
Lamichhane
Gerrit M
GM
Daubner
Judith
J
Thomas-Crusells
Sigrid D
SD
Auweter
Cristina
C
Manatschal
Keyunna S
KS
Austin
Oksana
O
Valniuk
Frédéric H-T
FH
Allain
David
D
Rueda
D017398:Alternative Splicing; D015153:Blotting, Western; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D038941:Polypyrimidine Tract-Binding Protein / Q000737:chemistry*; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D038941:Polypyrimidine Tract-Binding Protein; D012313:RNA