Expression proteomics of UPF1 knockdown in HeLa cells reveals autoregulation of hnRNP A2/B1 mediated by alternative splicing resulting in nonsense-mediated mRNA decay.
BACKGROUND
In addition to acting as an RNA quality control pathway, nonsense-mediated mRNA decay (NMD) plays roles in regulating normal gene expression. In particular, the extent to which alternative splicing is coupled to NMD and the roles of NMD in regulating uORF containing transcripts have been a matter of debate.


RESULTS
In order to achieve a greater understanding of NMD regulated gene expression we used 2D-DiGE proteomics technology to examine the changes in protein expression induced in HeLa cells by UPF1 knockdown. QPCR based validation of the corresponding mRNAs, in response to both UPF1 knockdown and cycloheximide treatment, identified 17 bona fide NMD targets. Most of these were associated with bioinformatically predicted NMD activating features, predominantly upstream open reading frames (uORFs). Strikingly, however, the majority of transcripts up-regulated by UPF1 knockdown were either insensitive to, or even down-regulated by, cycloheximide treatment. Furthermore, the mRNA abundance of several down-regulated proteins failed to change upon UPF1 knockdown, indicating that UPF1's role in regulating mRNA and protein abundance is more complex than previously appreciated. Among the bona fide NMD targets, we identified a highly conserved AS-NMD event within the 3' UTR of the HNRNPA2B1 gene. Overexpression of GFP tagged hnRNP A2 resulted in a decrease in endogenous hnRNP A2 and B1 mRNA with a concurrent increase in the NMD sensitive isoforms.


CONCLUSIONS
Despite the large number of changes in protein expression upon UPF1 knockdown, a relatively small fraction of them can be directly attributed to the action of NMD on the corresponding mRNA. From amongst these we have identified a conserved AS-NMD event within HNRNPA2B1 that appears to mediate autoregulation of HNRNPA2B1 expression levels.
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK.
Nicholas J
NJ
McGlincy
Lit-Yeen
LY
Tan
Nicodeme
N
Paul
Mihaela
M
Zavolan
Kathryn S
KS
Lilley
Christopher W J
CW
Smith
D020413:3' Untranslated Regions / Q000235:genetics; D017398:Alternative Splicing / Q000235:genetics*; D002352:Carrier Proteins / Q000378:metabolism; D018389:Codon, Nonsense / Q000235:genetics*; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics*; D006706:Homeostasis / Q000235:genetics*; D006801:Humans; D013058:Mass Spectrometry; D016366:Open Reading Frames / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics; D017346:Protein Serine-Threonine Kinases; D040901:Proteomics / Q000379:methods*; D020365:RNA Helicases; D020871:RNA Stability / Q000235:genetics*; D016372:RNA, Antisense / Q000378:metabolism; D016601:RNA-Binding Proteins; D015203:Reproducibility of Results; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D057825:Two-Dimensional Difference Gel Electrophoresis
D020413:3' Untranslated Regions; D002352:Carrier Proteins; D018389:Codon, Nonsense; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D020365:RNA Helicases; D016372:RNA, Antisense; D016601:RNA-Binding Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C468286:UPF2 protein, human; C439768:hnRNP A2; C118437:negative elongation factor; C514704:SMG1 protein, human; C101828:UPF1 protein, human