In vivo selection of kinase-responsive RNA elements controlling alternative splicing.
Alternative pre-mRNA splicing is often controlled by cell signals, for example, those activating the cAMP-dependent protein kinase (PKA) or the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV). We have shown that CaMKIV regulates alternative splicing through short CA repeats and hnRNP L. Here we use a splicing reporter that shows PKA/CaMKIV promotion of exon inclusion to select from exons containing random 13-nt sequences for RNA elements responsive to the kinases in cultured cells. This selection not only identified both PKA- and CaMKIV-responsive elements that are similar to the CaMKIV-responsive RNA element 1 (CaRRE1) or CA repeats, but also A-rich elements not previously known to respond to these kinases. Consistently, hnRNP L is identified as a factor binding the CA-rich elements. Analyses of the motifs in the highly responsive elements indicate that they are indeed critical for the kinase effect and are enriched in alternative exons. Interestingly, a CAAAAAA motif is sufficient for the PKA/CaMKIV-regulated splicing of the exon 16 of the CaMK kinase beta1 (CaMKK2) transcripts, implying a role of this motif in signaling cross-talk or feedback regulation between these kinases through alternative splicing. Therefore, these experiments identified a group of RNA elements responsive to PKA and CaMKIV from in vivo selection. This also provides an approach for selecting RNA elements similarly responsive to other cell signals controlling alternative splicing.
Li
Hongzhao
H
From the Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada.
Liu
Guodong
G
From the Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada.
Yu
Jiankun
J
From the Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada.
Cao
Wenguang
W
From the Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada.
Lobo
Vincent G
VG
From the Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada.
Xie
Jiuyong
J
From the Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada. Electronic address: xiej@cc.umanitoba.ca.
D017398:Alternative Splicing / Q000502:physiology*; D001483:Base Sequence; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4 / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000166:cytology; D016296:Mutagenesis; D012322:RNA Precursors / Q000502:physiology*; D020218:Response Elements / Q000235:genetics*
D012322:RNA Precursors; D017868:Cyclic AMP-Dependent Protein Kinases; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4