Alternative splicing and differential subcellular localization of the rat FGF antisense gene product.
BACKGROUND
GFG/NUDT is a nudix hydrolase originally identified as the product of the fibroblast growth factor-2 antisense (FGF-AS) gene. While the FGF-AS RNA has been implicated as an antisense regulator of FGF-2 expression, the expression and function of the encoded GFG protein is largely unknown. Alternative splicing of the primary FGF-AS mRNA transcript predicts multiple GFG isoforms in many species including rat. In the present study we focused on elucidating the expression and subcellular distribution of alternatively spliced rat GFG isoforms.


RESULTS
RT-PCR and immunohistochemistry revealed tissue-specific GFG mRNA isoform expression and subcellular distribution of GFG immunoreactivity in cytoplasm and nuclei of a wide range of normal rat tissues. FGF-2 and GFG immunoreactivity were co-localized in some, but not all, tissues examined. Computational analysis identified a mitochondrial targeting sequence (MTS) in the N-terminus of three previously described rGFG isoforms. Confocal laser scanning microscopy and subcellular fractionation analysis revealed that all rGFG isoforms bearing the MTS were specifically targeted to mitochondria whereas isoforms and deletion mutants lacking the MTS were localized in the cytoplasm and nucleus. Mutation and deletion analysis confirmed that the predicted MTS was necessary and sufficient for mitochondrial compartmentalization.


CONCLUSIONS
Previous findings strongly support a role for the FGF antisense RNA as a regulator of FGF2 expression. The present study demonstrates that the antisense RNA itself is translated, and that protein isoforms resulting form alternative RNA splicing are sorted to different subcellular compartments. FGF-2 and its antisense protein are co-expressed in many tissues and in some cases in the same cells. The strong conservation of sequence and genomic organization across animal species suggests important functional significance to the physical association of these transcript pairs.
Zhang
Shuo Cheng
SC
Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada. shuocheng.zhang@gmail.com
MacDonald
Kimberley A
KA
Baguma-Nibasheka
Mark
M
Geldenhuys
Laurette
L
Casson
Alan G
AG
Murphy
Paul R
PR
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D017931:DNA Primers / Q000235:genetics; D005346:Fibroblast Growth Factors / Q000235:genetics*; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D016372:RNA, Antisense / Q000235:genetics*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; C520403:NUDT6 protein, rat; D020033:Protein Isoforms; D016372:RNA, Antisense; D005346:Fibroblast Growth Factors