The mammalian exosome mediates the efficient degradation of mRNAs that contain AU-rich elements

Devi Mukherjee, Min Gao, J.Patrick O’Connor, Reinout Raijmakers, Ger Pruijn, Carol S. Lutz, and Jeffrey Wilusz

EMBO J. 2002 January 15; 21(1-2): 165–174.

Mike 8/11Exosome Journal Club

substrate specificity mechanisms

(determining substrate specificity)(exosome is recruited to substrate RNA )

RNAExosome

Nature Struct. Mol. Biol. 2004, 11, 121-126.

The enzyme and the control of eukaryotic mRNA turnover

1.ARE(AU-rich elements)-binding proteins TTP, RSAU and KSRP interact with exosome and recruit it to mRNA.

2.Exosome subunit (PM/Scl-75) binds ARE.

The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation.

1.Labile mRNAs that encode cytokine and immediate-early gene products often contain AU-rich sequences within their 3' untranslated region (UTR).

2.UUAUUUAUU is the minimal AU-rich motif that effectively destabilizes mRNA.

Mol Cell Biol. 1995 Apr;15(4):2219-30

AU binding proteins recruit the exosome to degrade ARE-containing mRNAs

Cell, Vol 107, 451-464, 16 November 2001

1.Mammalian exosome is required for rapid degradation of ARE-containing RNAs .

2. ARE recognition requires certain ARE binding proteins (KSRP,TTP, AUF1 and HuR) that can interact with the exosome and recruit it to unstable RNAs, thereby promoting their rapid degradation.

3’-5’exonucleolytic degradation is responsible for the decay of RNA substrates following deadenylation in HeLa cytoplasmic extracts.

AUG UAAPoly (G)

YeastmRNA turnover intermediates

exosome

deadenylation

3’-5’ exonucleolytic decay is the major, if not exclusive, pathway for turnover of mRNA following deadenylation in HeLa cytoplasmic extracts.

HeLa CellPhosphothioate

Exosomal proteins are required for 3’-5’ exonucleolytic decay in HeLa cytoplasmic extracts.

Immunodepletetion experiments

Exosome activity is regulated by AU-rich elements in mammalian cells.

PM-Scl75 protein interacts with RNAs that contain AU-rich instability elements.

ARE-TNF- ARE (34) ARE-GMCSF ARE(51)

PM-Scl75 protein interacts with RNAs that contain AU-rich instability elements.

1. 3’-5’exonucleolytic degradation is responsible for the decay of RNA substrates following deadenylation in HeLa cytoplasmic extracts.

2.Exosomal proteins are required for 3’- 5’exonucleolytic decay in HeLa cytoplasmic extracts.

3.Exosome activity is regulated by AU-rich elements in mammalian cells.

4.PM-Scl75 protein interacts with RNAs that contain AU-rich instability elements.

1.Loading the exosome complex onto mRNA is promoted by AREs in 3’- UTR.

2.Exosome may help promote mRNA deadenylation.

3.mRNA is degraded rapidly by exosome.

Summery

Model

1. 5’- 3’exonuclease activities exist in HeLa cytoplasmic extracts

AUG UAA A 60m7Gppp

Capped and polyadenylated RNA substrates are very stable in HeLa cytoplasmic extracts

substrate : SV-A60 RNA

AUG UAA A 70m7Gppp