RNA Degradation by the Exosome Is Promoted by a Nuclear Polyadenylation Complex John LaCava, Jonathan Houseley, Cosmin Saveanu, Elisabeth Petfalski, Elizabeth.

RNA Degradation by the Exosome Is Promoted by a Nuclear Polyadenylation Complex

John LaCava, Jonathan Houseley, Cosmin Saveanu, Elisabeth Petfalski, Elizabeth Thompson, Alain Jacquier, and David Tollervey

Cell, Vol. 121, 713–724, June 3, 2005

Chang-Hyun KimPTRM

Oct. 2, 2008

Introduction

• Exosome is multisubunit complex• 3’-5’ exonuclease activity• 15% of Exosome population is associated with

Rrp6 and restricted to the cell nucleus in yeast• Absence of Rrp6p impairs growth and leads to

Temperature sensitivity (Ts)

Introduction….continued• WhenRrp6p,1)accumulation of 3’-extended and polyadenylated

precursors to snoRNA and snRNA2)Polyadenylation of pre-RNA and rRNA,

modification defective tRNA• Polyadenylation of modification-defective tRNA

by Trf4p leads to degradation by the exosome

Introduction …..continued

• Purified exosome shows slow 3’-5’ exonuclease activity in vitro degradation intermediates ↑

• Individual components are active in vitro• Exosome is inactive in its default state to protect

cellular RNAs against inappropriate or nonspecific degradation

What is the activation cofactor for Exosome?

Authors’ Claim

• Mtr4p is associated with the TRAMP complex, which can activate the exosome for the degradation of RNA substrates in vitro and in vivo.

TRAMP complex = Trf4p/Air2p/Mfr4p

Two-hybrid analysis

• The two-hybrid system is a molecular genetic tool which facilitates the study of protein-protein interactions.

• If two proteins interact, then a reporter gene (the beta-galactosidase gene) is transcriptionally activateda colour reaction

• find proteins (prey) which interact with a protein you have already (bait).

Principle of the yeast two-hybrid system. The protein coding sequence of the bait protein is cloned into a vector containingthe DNA binding sequence (DBD-X (bait) fusion). The protein coding sequence of the prey protein is cloned into a vector that containssequences for transcription activation (AD-Y (prey) fusion). Both vectors must also contain the necessary elements for growth and protein expression in yeast. The recombinant vectors are introduced into the appropriate yeast strain. Only if proteins X and Y physically interact with one another are the DBD and AD brought together to reconstitute a functionally active factor that binds to upstream specific sequences of the reporter gene and activates expression.

Figure 1. (A) Regions of Trf4p and Trf5p identified in a two-hybrid screen with full-length Mtr4p as bait

Figure 1. (B) Coomassie blue-stained gel, showing large scale TAP purifications

*TRAMP complex coprecipitates each other and their association is not substrate RNAs dependent.

Figure 1. (C) Western analysis showing the salt sensitivity of coprecipitation of Mtr4p with Air2p-TAP,

Trf4p-TAP, and Trf5p-TAP

*TRAMP complex is salt labile.Nop1p = Nucleolar ribosome synthesis factor (Purification efficiency control)

Figure 2. TRAMP Polyadenylates an RNA Substrate

37 nt RNA substrate

500mM salt

Mtr4p-TAP

150mM salt

*TRAMP has polyadenylation activity and intact complex is required.

Figure 2. TRAMP Polyadenylates an RNA Substrate……..continued

S. pombe pre-tRNA

*TRAMP show a distributive mode of poly(A) addition, which might be an important feature of a polymerase that is associated with a degradative activity. Exosome??

Figure 3. The TRAMP Complex Stimulates RNA Degradation by the Exosome

*Combination of the exosome and TRAMP complex enhances the processivity of the exosome.

Figure 3. (B) The 110 nt pre-tRNA substrate was incubated with the exosome and/or TRAMP as indicated

Figure 3. ….continuedPhysiological condition(150mM salt)

500 mM salt

*Intact TRAMP complex is required for tRNA degradation by exosome.

Figure 4. Stimulation of the Exosome Requires the Intact TRAMP Complex

100 mM Salt

Phosphoimager quantification of input RNA remaining with time

Figure 4. …..continued

*Exosome activity requires intact TRAMP complex and is polyadenylation independent Cordycepin triphosphate = non-extendible ATP analog

110nt

Summary for in vitro experiment• TRAMP complex coprecipitates each other and their

association is not substrate RNAs dependent• TRAMP complex is salt labile.• TRAMP has distributive mode of polyadenylation activity and

intact complex is required• Combination of the exosome and TRAMP complex enhances

the processivity of the exosome.• Intact TRAMP complex is required for substrate RNA

degradation by exosome• Exosome activity requires intact TRAMP complex and is

polyadenylation independent

Figure 5. The TRAMP Complex is Required for Normal RNA Processing and Degradation In Vivo

Thioredoxin peroxidase(loading control)

*TRAMP mutants have impaired RNA degradation.

Figure 5. (E)(F) alterations in U5 isoforms

Figure 5. (G) Quantification of alterations in U5 isoforms, with the wild-type ratio set as 1

Figure 6. Polyadenylated RNAs Are Accumulated in Strains Lacking the Exosome Component Rrp6p but Not in TRAMP Mutants

Figure 6. Polyadenylated RNAs Are Accumulated in Strains Lacking the Exosome Component Rrp6p but Not in TRAMP Mutants ……continued

Figure 6. Polyadenylated RNAs Are Accumulated in Strains Lacking the Exosome Component Rrp6p but Not in TRAMP Mutants ……continued

Model for the Roles of the TRAMP Complex in RNA Degradation

Pros and Cons

• Identified and characterized TRAMP complex• TRAMP as an Exosome activation cofactor

• The role of Trf5p needs to be further investigated

• The role polyadenylation by Trf4p on exosome activation is not clear