Drosophila maternal Hsp83 mRNA destabilization is directed by multiple SMAUG recognition elements in the open reading frame.
SMAUG (SMG) is an RNA-binding protein that functions as a key component of a transcript degradation pathway that eliminates maternal mRNAs in the bulk cytoplasm of activated Drosophila melanogaster eggs. We previously showed that SMG destabilizes maternal Hsp83 mRNA by recruiting the CCR4-NOT deadenylase to trigger decay; however, the cis-acting elements through which this was accomplished were unknown. Here we show that Hsp83 transcript degradation is regulated by a major element, the Hsp83 mRNA instability element (HIE), which maps to a 615-nucleotide region of the open reading frame (ORF). The HIE is sufficient for association of a transgenic mRNA with SMG protein as well as for SMG-dependent destabilization. Although the Hsp83 mRNA is translated in the early embryo, we show that translation of the mRNA is not necessary for destabilization; indeed, the HIE functions even when located in an mRNA's 3' untranslated region. The Hsp83 mRNA contains eight predicted SMG recognition elements (SREs); all map to the ORF, and six reside within the HIE. Mutation of a single amino acid residue that is essential for SMG's interaction with SREs stabilizes endogenous Hsp83 transcripts. Furthermore, simultaneous mutation of all eight predicted SREs also results in transcript stabilization. A plausible model is that the multiple, widely distributed SREs in the ORF enable some SMG molecules to remain bound to the mRNA despite ribosome transit through any individual SRE. Thus, SMG can recruit the CCR4-NOT deadenylase to trigger Hsp83 mRNA degradation despite the fact that it is being translated.
Semotok
Jennifer L
JL
Department of Molecular Genetics, University of Toronto, Medical Sciences Building, Room 4384D, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.
Luo
Hua
H
Cooperstock
Ramona L
RL
Karaiskakis
Angelo
A
Vari
Heli K
HK
Smibert
Craig A
CA
Lipshitz
Howard D
HD
D000818:Animals; D001483:Base Sequence; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D020871:RNA Stability; D023121:RNA, Messenger, Stored / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes
D029721:Drosophila Proteins; D006360:Heat-Shock Proteins; C089272:Hsp83 protein, Drosophila; D023121:RNA, Messenger, Stored; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C402587:smg protein, Drosophila
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