Eukaryotic mRNA Degradation Reading: General mRNA decay: Mitchell and Tollervey Current opinion in Genetics and Dev. 2000 10:193-198. NMD: Lykke-Andersen et al Science 2001 293:1836-1839. RNAi: Zamore Nature Structural Biology 2001 8 : 746 - 750.

outline Cis-acting mRNA elements Pathways of decay Deadenylation Subsequent 5’-decay Subsequent 3’-decay Nonsense-mediated decay RNAi

Elements of an mRNA that affect its stability coding region determinant stem loop exon-exon junction AUG UAG ARE 5’cap orf AAAAAAA 3’-UTR 5’-UTR premature termination codon 5’-cap: protection against 5’-exonuclease Stem loop: inhibition of translation can stabilize mRNA Coding region determinant: can mask other mRNA elements to stabilize untranslated mRNA Premature termination codon: target transcript for nonsense-mediated decay (NMD) Exon-exon junction: binding site for nuclear shuttling proteins - determinant for NMD AU-rich element (ARE): binding site for destabilizign or stabilizing proteins

Pathways of general mRNA decay in yeast

Decay of stable and unstable mRNAs in yeast initiates with deadenylation followed by degradation 5’ to 3’ deadenylation deadenylation Position of poly(A) minus mRNA. Sample was treated with oligo dT and RNaseH Transcriptional pulse-chase of PGK1 (stable) and MFA2 (unstable) in yeast. Tracts of 18 consecutive guanosines (pG) were engineered into the mRNAs for strong secondary structures to block exonuclease degradation. From Decker and Parker Genes & Dev. 7:1632-1643 (1993). In mammalian cells, pG does not inhibit degradation and so the in vivo polarity of decay is not known.

Deadenylation in yeast requires Ccr4p in vivo and is blocked by PAB in vitro eIF4G Deadenylase eIF4E PAB PAB AAAAAA Pab1p inhibits Ccr4p deadenylase activity in vitro. Analysis of deadenylation activity in Flag-Ccr4p purified fractions with addition of increasing amounts of purified Pab1p. Purified Pab1p was added to each time course in molar amounts relative to Flag-Ccr4p as indicated. Numbers above the lanes indicate time points taken after addition of substrate to the reaction. The asterisk indicates the position of the radiolabeled phosphate. From Tucker et al EMBO,( 2002) 21,1427-1436.

Deadenylation AU-rich elements (AREs) in the 3’-UTR of an mRNA destabilize the RNA. Different classes of AREs result in different kinetics of deadenylation

Deadenylation in mammalian extracts requires DAN DAN (or PARN) activity is stimulated by cap but access to the polyA tail by DAN is blocked by the presence of translation factors as well as RNA binding proteins such as HuR. Other RNA binding proteins such as ARE-binding hnRNP D destabilize mRNAs possibly by disrupting PAB binding. Transient dissociation of PAB during translation or by the post termination ribosome may allow access for DAN. However, since deadenylation has not been recapitulated in extracts, the mechanism remains unclear. Also note that DAN is not related to yeast Ccr4.

5’-degradation in yeast Following deadenylation 5’to 3’degradation requires access to the cap by a decapping enzyme. eIF4G PAB PAB eIF4E AAAAAA Dcp1 Competition between Dcp1 and eIF4E. Kd of eIF4E-cap >> Km of Dcp1-cap. However, interaction of eIF4G strengthens eIF4E interaction with cap and eIF4G interaction with PAB may help stabilize mRNP. Mutations in PAB, eIF4G, eIF4E or eIF3 lead to increased decapping by Dcp1. A related decappng activity in human cells has not been identified.

In yeast remodeling of the mRNP preceding degradation requires Pat1 and the Lsm complex eIF4G eIF4E PAB PAB Pat1 AAAAAA Remodeling of mRNP eIF4G eIF4E PAB PAB Lsm complex Pat1 Dcp1 AAAAAA Pat1p is found in mRNP complexes containing eIF4E, eIF4F, Pab or Lsm1-7, and Dcp1, suggesting a remodeling of the mRNP to recruit Dcp1 via the Lsm complex. See Tharun and Parker Mol Cell 2001 8:1075-1083.

In mammalian extracts, 3’-decay is the predominant pathway after deadenylation -this is a minor pathway in vivo in yeast recruited to ARE via ARE binding protein ARE binding protein exosome binds ARE directly 5’-cap ARE The exosome is reported to bind AREs directly. It is also reported to bind ARE binding proteins including AUF1 suggesting indirect recruitment. The exosome does not bind HuR, so HuR could protect ARE-containing mRNAs from 3’-decay.

Purified exosome is inactive and apparently requires adapters (helicases) for substrate recognition Models for the activation of the exosome. a, Proteasome model. Access to the active sites of the exonucleases in the exosome is regulated by the RNA helicase Mtr4p or Ski2p (blue oval). Displacement or reorganization of the helicase occurs upon interaction with the RNP substrate. ATP hydrolysis by the activated RNA helicase unfolds the RNA structure and channels the free 3' end into the lumen of the exosome. b, Allosteric model. The RNA helicase interacts directly with the RNP substrate, recognizing specific marker proteins. The ATPase activity of the RNA helicase allows the exosome to be remodeled into an appropriately active form. The helicase may interact with the RNP substrate, either associated with the exosome or in its absence; for simplicity only the latter case is shown. From Mitchell Tollervey (2000) Nature Structural Biology 7, 843 – 846.

Nonsense-mediated decay Premature stop codons can target a transcript for rapid, deadenylation-independent decay ORF 5’ 3’-UTR stop Normal stability AAAAAAAA NMD ORF 5’ 3’-UTR stop AAAAAAAA Premature stop preCYH2 (stop codon in intron) nonsense-PGK1 CYH2 MFA2

Model for NMD in mammalian cells From Lykke-Andersen Curr Biol. 2001 11(3):R88-91.

NMD can be induced by tethering some exon-junction proteins to mRNA hUpf3 or RNPS1 (Y14) MS2 NMD 5’ b-globin 3’-UTR stop UAA hUpf3 or RNPS1 (Y14) MS2 NMD 5’ b-globin stop

In human cells cytoplasmic NMD may occur during first (pioneer) round of translation as a transcript is exported from the nucleus From Ishigaki et al Cell, 106:607-617 2001.

RNA interference (RNAi) From Zamore Nature Structural Biology 2001 8 : 746 - 750.

Possible mechanism of amplification of siRNAs using a RNA-directed RNA polymerase (RdRP) Adapted from Lipardi et al Cell. 2001 Nov 2;107(3):297-307.