2011 MECHANISMS OF CYTOPLASMIC mRNA TURNOVER IN EUKARYOTES

2011 MECHANISMS OF CYTOPLASMIC mRNA TURNOVER IN EUKARYOTES
Kim Keeling, Ph.D. Room 456, Bevill Bldg The main focus of today's lecture will be "mechanisms of cytoplasmic mRNA turnover in eukaryotes". I will mainly focusing on cytoplasmic mRNA turnover. Much of the machinery for cytoplasmic mRNA turnover is also used, in addition with other nuclear factors, to degrade mRNAs in the nucleus as well and I will try to point out the similarities as I go through the lecture.

Lecture Overview 1. General mRNA turnover pathways
- Play key role in controlling basal gene expression levels 2. Aberrant RNA turnover pathways - Recognize and degrade aberrant mRNAs or mRNAs that are translated inefficiently - Increases the quality control of mRNA biogenesis and gene expression 3. Specialized mRNA turnover pathways - mRNA turnover acts as a site for regulation in response to specific signals (hormones, cell cycle, viral infection, differentiation, nutrient availability, stress) - AU-rich element mediated (ARE) 4. Specialized sites of cytoplasmic mRNA turnover - Processing bodies (P bodies) - Exosome granules for ARE-mediated mRNA turnover I will go over 4 main points in this lecture. 1) the general mRNA turnover pathways used to degrade all mRNAs; 2) mRNA pathways that degrade aberrant mRNAs; 3) specialized mRNA pathways; 4) specialized sites of mRNA turnover.

Determining mRNA Stability Experimentally
10 20 30 40 50 60 70 80 90 100 t1/2 = tpa1 24 min wild type 12 min % ACT1 remaining Time (m) time (m) WT tpa1∆ inhibit expression Northern blot of ACT1 mRNA 1. Shut off transcription: thiolutin, actinomycin D, promoters that can be repressed (such as copper or tetracycline regulated promoters). 2. Isolate mRNA at several time points after transcriptional shut-off. 3. Determine the percent of mRNA remaining at each time point after transcriptional shut-off. 4. Graph the level of RNA remaining for each time point on a semi-log graph. The slope = the rate of mRNA decay. The time at which half of the mRNA is degraded is the half-life of the mRNA. Let's first talk about how we monitor the stability of mRNAs experimentally. The steady level of a mRNA is determined by the combination of how much is produced by transcription together with the rate the mRNA is degraded. In order to focus solely on the rate of degradation, we must first inhibit the transcription of the mRNA. This can be done by inhibiting the polymerase with drugs such as actinomycin D in mammalian cells and thiolution in yeast cells. Another way to inhibit transcription is to place the gene of interest under the control of a repressible promoter such as tetracycline or copper. After transcription is inhibited, the mRNA is harvested at numerous time points and purified and isolated by gel electrophoresis. The RNA can then be transferred to a membrane and hybridized with a gene-specific probe. The amount of RNA that remains at each time point is plotted against time on a semi-log graph to determine the rate that the mRNA is degraded (slope). The stability of a mRNA is often expressed as the half-life = the time at which 50% of the mRNA remains. Describe yeast experiment. Keeling et al. (2006) Mol Cell Biol 26:5237

mRNAs Are Modified During Nuclear
Processing to Promote Cytoplasmic Stability Nucleus Cytoplasm 1. Addition of 5’ cap and the CAP binding protein complex 2. Addition of the 3’ poly(A) tail and multiple poly(A) binding proteins 3. Circularization of mRNA by bridging the 5’ CAP and the 3’ poly(A) tail via eIF4G What are some factors that control mRNA stability? Anyone that works with RNA experimentally knows that special care must be taken to keep the RNA from being degraded. Multiple RNA nucleases exist that degrade RNA. In order for the mRNA to remain stable enough to be translated, mRNA must be modified during its transcription. First, a 7-methyl guanosine is added to the 5’ end of the mRNA and it is bound by proteins called CAP binding proteins in both the nucleus and the cytoplasm. Multiple adenosines, called the poly(A) tail are added to the the 3’ end of the mRNA and this is also bound by proteins called poly(A) binding proteins. Multiple Pabs bind the poly(A) tail to protect it against degradation. After the mRNA is exported to the cytoplasm the 5’ CAP and the 3’ poly(A) tail are bridged together via an mutual interaction with eIF4G. This closed loop structure essentially circularizes the mRNA which further stabilizes the mRNA and enhances translation. Goldstrohm & Wickens (2008) Nat Rev Mol Cell Biol 9:337

1) General mRNA turnover pathways
We will first talk about the pathways that are used to degrade mRNAs.

The Major Eukaryotic mRNA Turnover Pathways
There are two general pathways to degrade mRNAs that have reached their lifespan. As you might imagine, these pathways focus on accessing the ends of mRNAs to be degraded by exonucleases. The first pathway is the 5’ to 3’ pathway. In this pathway, the mRNA is first deadenylated (the poly(A) tail is removed), then decapped and degraded in the 5’ to 3’ direction. This pathway is the dominant pathway in yeast. The second pathway is the 3’ to 5’ pathway. The first step in this pathway is also deadenylation. However, after deadenylation, the mRNA is not decapped, rather, it is degraded in the 3’ to 5’ direction by the cytoplasmic exosome and the remaining cap is degraded by the scavenger decapping enzyme. This pathway is the dominant pathway in mammalian cells. CAP hydrolysis Biochem. Soc. Trans. (2006) 34:35

Deadenylation is a common step that must occur first in both mRNA turnover pathways. CAP hydrolysis Biochem. Soc. Trans. (2006) 34:35

Mammalian Deadenylation Occurs in Two Phases
PAN2 complex Slow CCR4 complex Fast Two deadenylase complexes appear to work in a sequential manner to remove the poly(A) tail. The PAN complex, which is activated by Pab1, acts during the first phase of deadenylation which is slow and distributive so that as Pab1 molecules fall off the mRNA, the PAN complex slowly chews off a few of the exposed adenosines and then falls off. When the poly(A) tail is shorted to a certain length, the second phase of deadenylation is carried out by the ccr4/pop2 complex which can bind to the poly(A) tail and remove the remaining adenosines in a rapid, processive manner. Muhlemann (2005) Nat Struct Mol Biol 12:1024

Deadenylase Machinery
1) PAN complex: - First phase - Slow, distributive - Stimulated by PAB - Also trims poly(A) tails in nucleus 2) CCR4 complex: - Second phase - Fast, processive - Inhibited by PAB - Independent function as a transcription factor Let's talk about the deadenylase machinery in more detail. The PAN complex is composed of Pan2, the catalytic subunit, and Pan3, a regulatory subunit. Stimulated by PABP, also functions as deadenylase in nuclease to trim poly(A) tails to the correct size. CCRF is composed of the Ccr4 catalytic subunit, the Pop2 regulatory subunit, and other factors. In addition to acting as a deadenylase in the nuclease, it also has an independent function as a transcription factor mediated by accessory proteins. PARN is a deadenylase used for specialized deadenylation that I will talk about later in the lecture. 3) PARN complex: - Used for specialized rapid deadenylation (ARE-mediated decay) in mammalian cells Parker & Song (2004) Nat Struct Mol Biol 11:121

Determining Deadenylation Rates Experimentally
RNase H Northern blot RNase H T + oligo dT  poly(A)- PTETOFF AAAAAAAAAA Probe DNA oligo Northern blot Control RNA Transcriptional shut off How do we experimentally monitor the rate of deadenylation? We perform the same experiment as we did to measure mRNA turnover rates with the addition of a couple of steps. In order to better resolve just the rate of poly(A) tail removal we will cleave off the majority of the mRNA by incubating the isolated mRNA with a DNA oligo that is complementary to a region within the C-terminus of the mRNA and treated the RNA with RNase H. RNase H cleaves DNA:RNA hybrids. This allows the poly(A) tail region to be resolved using electrophoresis and then northern blotted using a probe within the 3’ UTR. An oligo dT is added to an RNA sample during RNase H treatment to determine the size of the mRNA without a poly(A) tail. Deadenylation occurs in 2 phases: an initial slow, synchronous phase followed by a fast, asynchronous phase when the poly(A) tail has reached about 100 adenosine. The second phase of deadenylation is followed by degradation of the body of the mRNA. slow synchronous fast asynchronous PAN complex CCR4 complex Yamashita et al. (2005) Nat Struct Mol Biol 12:1054

PAN and CCR4 Sequentially Deadenylate mRNAs
Enhanced 1st phase Enhanced 2nd phase No difference The Pan and Ccr4 deadenylases work in sequence. In this experiment, Yamashita et al. analyzed the rate of deadenylation in mammalian cells that overexpressed either WT or mutant components of the deadenyase machinery. They found that if they overexpressed WT pan2, they could see enhanced deadenylation during the slow, synchronous phase, while mutant pan2 showed a delayed initial deadenylation. When WT ccr4 was overexpressed, the second fast, asynchronous phase of deadenylation was enhanced, while expression of mutant ccr4 delayed the second phase of deadenylation. PARN did not change the deadenylation rates of this transcript. Delayed 1st phase Delayed 2nd phase No difference Yamashita et al. (2005) Nat Struct Mol Biol 12:1054

Deadenylation Is Coupled to Translation Termination
Hosoda et al (2003) JBC 278:38287 The ability of poly(A) binding protein to assemble onto the poly(A) tail is crucial in preventing deadenylation of a mRNA and subsequent mRNA decay. It has been found that the process of translation termination can influence the ability of Pab to remain associated with the poly(A) tail and represents a way that the stability of mRNAs might be controlled. Translation termination occurs when a stop codon enters the ribosomal A site. The stop codon is recognized by proteins called release factors. There are two release factors. eRF1 binds to the A site and mediates release of the nascent polypeptide. eRF3 is a GTPase that binds to eRF1 and enhances it function. eRF3 also interacts with Pab1. The interaction of between eRF3 and Pab1 diminishes the ability of Pab1 to oligomerize on the poly(A) causing Pab1 to fall off the poly(A) tail making it susceptible to deadenylation. - eRF3 is a GTPase that mediates translation termination. - The N-terminal domain of eRF3 interacts with the Pab1p C-terminal domain. - This interaction inhibits the ability of Pab1p molecules to oligomerize efficiently on the poly(A) tail. This increases the exposure of the poly(A) tail to deadenylases and turnover.

eRF3 Binds to Pab1p and Inhibits Its Oligomerization
Hoshino et al. (1999) Biochem (Mosc) 64:1367 In this gel shift assay, Hoshino et al, incubated a radiolabeled RNA with increasing concentrations of Pab. As more Pab1 is added, additional Pab1 molecules bind to the RNA which causes a gel-shift up due to the increased molecular weight. When the N-terminal domain of eRF3 (the domain which interacts with Pab) is added in addition to the Pab1, less Pab1 is able to oligomerize onto the mRNA suggesting that eRF3 prevents Pab1 molecules from interacting together on the poly(A) tail. - Gel shift assay: radio-labeled RNA is incubated with increasing amounts of Pab1p protein. - A shift in the mobility of the RNA indicates Pab1p protein binding. - Addition of the eRF3 N-terminal domain to the binding assay inhibits the ability of Pab1p to oligomerize on the RNA.

The eRF3-Mediated Inhibition of Pab1p Results
in Deadenylaton and mRNA Turnover Hosoda et al (2003) JBC 278:38287 Hosada et al. show in this experiment that expressing eRF3 that lacks its N-terminal domain leads to longer poly(A) tail lengths. These are RNase H northern blots of steady state MFA2 mRNA. They also show that this mutant eRF3 inhibits the turnover rate of mRNAs. This data suggests that under normal conditions, WT eRF3 promotes deadenylation and mRNA turnover by interfering with the ability of Pab1 to associate on the poly(A) tail. - Expression of a mutant eRF3 where the N-terminal domain is deleted so that it can no longer interact with Pab1p results in longer poly(A) tail length. This mutant also leads to an increased mRNA half-life = decreased turnover rate. - This suggests that normally, eRF3 mediates mRNA turnover by decreasing Pab1p oligomerization on poly(A) tails which leads to deadenylation and mRNA decay. This represents one way to mediate general mRNA decay.

Pan2, Ccr4 For the 5' to 3' turnover pathway, the mRNA is then decapped. CAP hydrolysis Biochem. Soc. Trans. (2006) 34:35

Decapping Enzymes Dcp1 - Dcp2p = catalytic subunit
Yeast interactions Human interactions - Dcp2p = catalytic subunit - Dcp1p = stimulates Dcp2p - Hedls = also needed to stimulate Dcp2p activity in mammals - In yeast, Dcp1p and Dcp2p can directly interact with each other to stimulate decapping - Dcp2p is differentially expressed in mammalian tissues - Another decapping enzyme was identified in the cytoplasm of mammalian cells called Nudt16. No ortholog found in yeast, C. elegans or Drosophila. - Nudt16 and Dcp2 appear to each work on subsets of cellular transcripts. Hedls Dcp2 Simon et al. (2006) TIBS 31:241 The protein Dcp2 is the catalytic subunit of the decapping complex. Dcp1 is a regulatory subunit. In yeast these two proteins are can directly interact and stimulate decapping. In mammalian cells, another protein is required for Dcp2 and Dcp1 to interact called Hedls. Recently, an additional decapping enzyme called Nudt16 was identified in mammals. The decapping enzymes appear to each work on subsets of cellular transcripts. Nudt16 Taylor & Peculis (2008) NARS 36:6021

Control of the Decapping Reaction
1. Poly(A) tail status - Poly(A) binding protein inhibits decapping by physically interacting with the 5’ cap complex (via eIF4G) to stabilize the cap and enhance translation. 2. CAP status - The CAP protein complex must dissociate from the cap prior to decapping. 3. Translation status - A transition must take place from a translation competent state to a state where translation does not take place (change in mRNP structure). 4. Assembly of the decapping complex - Specific factors are required for the localization, assembly, and activation of the decapping complex. The decapping step is a highly regulated one and can be controlled by a number of factors. 1) The poly(A) tail status can influence decapping. 2) The translation status controls decapping. 3) A transition must take place from a translation-competent state to a translation-incompetent state. For example the CAP binding complex must be removed. 4) Specific factors are needed to localize, assemble, and activate the decapping enzymes.

Decapping Activators Pat1p
- Interacts with both poly(A) [+] and poly(A) [-] transcripts - Seeds decapping complex onto mRNA - Interacts with Dcp1p, Dcp2p, Lsm1-7p, Dhh1p, Xrn1p - Along with the Lsm complex, represses translation Lsm1-7p - 7 ring complex interacts with poly(A) [-] mRNA - Facilitates the assembly of the decapping complex in cytoplasmic and nuclear decapping - Interacts with Dcp1p, Dcp2p, Dhh1p, Pat1p, Xrn1p Dhh1p - ATP dependent helicase - Binds directly to mRNA substrate - Interacts with Dcp1p, Dcp2p, Lsm1-7p, Ccr4p, Pop2p, Edc1p, Edc2p Edc1/Edc2 - Enhances decapping activity - Interacts with Dcp1 and Dcp2 - Enhances alterations in mRNA stability in response to nutrient changes Parker & Song (2004) Nat Struct Mol Biol 11:121 Various

Pan2, Ccr4 Dcp2, Nudt16 The final step in the 5' to 3' pathway is degradation of the mRNA by the exonuclease Xrn1. CAP hydrolysis Biochem. Soc. Trans. (2006) 34:35

Pan2, Ccr4 Dcp2, Nudt16 The nuclear 5' to 3' exonuclease = Xrn2 in human cells; yeast homologue = Rat1. CAP hydrolysis Xrn2/Rat1 = nuclear exonuclease Biochem. Soc. Trans. (2006) 34:35

Pan2, Ccr4 The second pathway is the 3’ to 5’ pathway. The first step in this pathway is also deadenylation. However, after deadenylation, the mRNA is not decapped, rather, it is degraded in the 3’ to 5’ direction by the cytoplasmic exosome and the remaining cap is degraded by the scavenger decapping enzyme. This pathway is the dominant pathway in mammalian cells. CAP hydrolysis Biochem. Soc. Trans. (2006) 34:35

Exosome Components Core Subunits: all essential Rrp4p Rrp40p Csl4p
Rrp41p/Ski6p Rrp42p Rrp43p Rrp44p/Dis3p Rrp45p Rrp46p Mtr3p Associated factors: Mtr4p essential ATP-dependent helicase Ski2p non-essential ATP-dependent helicase Ski3p non-essential TPR repeat Ski8p non-essential WD repeat Ski7p non-essential GTPase Nuclear subunits: Rrp6p non-essential nuclear subunit The exosome is a complex of at least 10 subunits which produce a ring of exonucleases that degrade the mRNA. The exosome can not bind to the mRNA directly, but requires adaptor molecules known as the ski complex to be able to efficiently associate with its mRNA substrate in the cytoplasm. There is also a nuclear exosome that includes all of the listed core subunits with the addition of a nuclear subunit Rrp6. The adaptor molecule for RNA degradation in the nucleus is known as the TRAMP complex. Parker & Song (2004) Nat Struct Mol Biol 11:121

The Exosome Gains Access to mRNAs for
Degradation Via Ski7p and the SKI Complex The SKI complex composed by ski2, ski3, and ski8 bind to the mRNA. Ski7p bridges and interaction between the SKI complex and the exosome to allow degradation of the mRNA. - Ski7p bridges an interaction between the mRNA-bound SKI complex and the exosome. - In the nucleus, a group of factors called the TRAMP complex is the bridge between mRNAs and the exosome. Araki et al. (2001) EMBO J 20:4684

Pan2, Ccr4 Dcp2, Nudt16 The final step in mRNA degradation is CAP hydrolysis. CAP hydrolysis Biochem. Soc. Trans. (2006) 34:35

Final Step of mRNA Decay: Degrading the 5’ Cap
The final step in the 3’ to 5’ pathway is the degrade the 5’ cap. The scavenger decapping enzyme accomplishes this final step. - The scavenger decapping enzyme hydrolyzes di- and tri-phosphorylated CAPs to the mono form. - Substrates are less than 10 nucleotides in length. Parker & Song (2004) Nat Struct Mol Biol 11:121

Summary of the General Eukaryotic
mRNA Decay Pathways Summary of general mRNA pathways. Meyer et al. (2004) Crit Rev Biochem Mol Biol 39:197

Nuclear mRNA Decay Also Utilizes Components of the 5'  3' and 3' 5' mRNA Turnover Pathways
Moore (2002) Cell 108:431 TRAMP Xrn2p mRNAs that are not processed properly are subject to turnover in the nuclear to prevent their escape from the nucleus. Nuclear RNAs are also deadenylated, decapped and degraded by the 5' to 3' and 3' to 5' pathways. The nucleus 5' to 3' exonuclease is called Xrn2 in human cells and Rat1 in yeast. For the 3' to 5' pathway, the TRAMP complex recruits the exosome to the mRNA for degradation. Nuclear Yes Xrn2p/Rat1p TRAMP

2) Aberrant mRNA Decay Pathways
A. Nonsense-mediated mRNA decay (NMD) - Degrades mRNAs with premature stop codons B. Nonstop mRNA decay (NSD) - Degrades mRNAs without a stop codon C. No-go mRNA decay (NGD) - Degrades mRNAs that have a stalled ribosome D. Ribosome extension-mediated decay (REMD) - Degrades mRNAs where ribosome translates past the stop codon and into the 3’ UTR Now we are going to talk about the mRNA decay pathways that degrade aberrant mRNAs. The four listed are surveillance pathways that provide quality control of mRNAs and translation status.

Nonsense-Mediated mRNA Decay
- Specialized pathway that degrades mRNAs that contain premature translation termination signals - Protects the cell from translating mRNAs that might produce truncated peptides that could lead to harmful dominant negative effects - Occurs in all eukaryotes. - 30% of disease-generating mutations result in premature stop codons - Up to 10-20% of the transcriptome is regulated by NMD - PTC-containing transcripts caused by point mutations, frameshift mutations, mRNAs with faulty alternative splicing, pre-mRNAs that escape nuclear retention, mRNAs that contain upstream open reading frames, mRNAs that carry introns in 3´ untranslated regions, or mRNAs with long 3´ untranslated regions Czapllinski et al. (1999) Bioeassay 21:685 The first pathway we will talk about is the nonsense mediated mRNA decay pathway = NMD. This pathway degrades mRNAs that contain premature stop mutations. As shown here the half-life of a mRNA with a premature stop codon is rapidly reduced compared to a WT mRNA.

Current NMD Models Splicing-independent NMD Splicing-dependent NMD
Normal Termination Splicing-dependent NMD Aberrant Termination Currently there are two models that explain how NMD might work. In mammals, mRNAs are spliced and some of the factors required for mammalian NMD associate with the mRNA during splicing. However, in organisms such as yeast, on about 5% of mRNAs undergo splicing, yet NMD works in both organisms. Therefore, an alternative form of NMD has been proposed for splicing-independent NMD. The splicing independent NMD pathway is dependent upon the efficiency of translation termination that is influenced by the distance between a stop codon and the Pab protein. Wen & Brogna (2008) Biochem Soc Trans 36:514

Splicing-Dependent NMD
Let’s talk about the splicing-dependent model of NMD first. In this model some NMD factors associate with the mRNA in the nucleus as it is being spliced. These factors bind to the mRNA about 24 nts upstream of each exon-exon junction and is called the EJC or exon junction complex. Some of these factors remain associated with the mRNA as it is exported to the cytoplasm. In the cytoplasm, when the mRNA undergoes the first round of translation called the pioneer round, the ribosome removes the EJC during translation. If there is no PTC, the ribosome is able to remove all the EJCs and remodel the mRNA for subsequent rounds of steady-state translation. However, if a PTC is present in the mRNA, translation termination will occur at the PTC. If an EJC remains on the mRNA, the mRNA can not be fully remodeled and this signals that the mRNA is aberrant and it is degraded.

NMD Factors Associate With the EJC
core NMD components There are a number of factors required for NMD that associate with the EJC in the nucleus as shown here. However, there are 3 core NMD factors that are conserved throughout eukaryotes. These include Upf3, which associated with the EJC, Upf2, which resides in the perinuclear space and binds to Upf3 as the mRNA is exported from the nucleus to the cytoplasm. Upf1 is a cytoplasmic NMD factor that associates with the mRNA during translation. Core NMD Components: UPF3: associates with the EJC in the nucleus UPF2: perinuclear and binds to Upf3 as the mRNA is exported UPF1: associates at the stop codons in mRNAs during translation

Regulation of NMD by Upf1p Phosphorylation
1. The SURF complex composed of SMG1, UPF1, and the eRF1 and eRF3 release factors forms. This complex recognizes stop codons. 2. If the SURF complex interacts with a downstream EJC complex that includes UPF2, UPF3, and Y14 (the DECID complex), then SMG-1 phosphorylates UPF1. This marks the stop codon as premature. 3. UPF1 phosphorylation induces a change in the mRNP structure that recruits SMG5, SMG6, SMG7, and the PP2A phosphatase to dephosphorylate UPF1. This signals for the mRNA to be degraded. Recently, it has been discovered that not all mRNAs that are subject to NMD require all of the NMD factors, some are Upf2-independent and others may be Upf3-independenct. However, all NMD required Upf1. The modification of Upf1 is crucial to NMD. Upf1 forms a complex with eRF1, eRF3 and the SMG-1 kinase…. Dephosphorylation of Upf1 can recruit machinery from multiple mRNA degradation pathways to degrade the mRNA. Kashima et al. (2006) Gene Dev 20:355

Aberrant Translation Termination Mediates NMD
As we mentioned earlier, NMD occurs in organisms where splicing of mRNAs are very rare. An alternative model for NMD has been devised to explain how NMD might occur without splicing. In this model, the distinction between normal termination and aberrant termination determines whether a mRNA is destined for NMD. Rather than downstream effectors such as an EJC determining whether a stop codon is premature, the spatial distance between the stop codon and the factors bound to the 3’ UTR determine if termination is aberrant or not. If the factors bound to the stop codon can interact with components of the 3’ UTR, (eRF3 binds to Pab1) termination is normal, however, if the distance between the stop codon (PTC) does not allow efficient interactions with factors bound to the 3’ UTR, (eRF3 binds to Upf1) termination is aberrant and the mRNA is degraded. It has been proposed that the EJC is a way to improve the efficiency of the aberrant termination model in organisms that rely heavily on splicing for genetic diversity such as mammals. Muhlemann (2008) Biochem Soc Trans 36:497

Nonstop mRNA Decay - Pathway to degrade mRNAs
that do not contain any stop codons - Typically caused by the presence of cryptic poly(A) addition sites that leads to polyadenylation of transcripts upstream of the termination signal - Utilizes Ski7p, the SKI complex, and the exosome to degrade nonstop messages The nonstop mRNA decay pathway (NSD) degrades mRNAs that do not contain stop codons. The ribosome translates down into the 3’ UTR until it reaches the end of the mRNA with the A site of the ribosome empty. Ski7 binds to the ribosome and recruits the SKI complex and the exosome to degrade the message. Maquat (2002) Science 295:2221

No Go mRNA Decay - Degrades messages with translation
elongation stalls (i.e. hairpin structures, pseudoknots, rare codons) - Requires two factors that bind to the A site of the stalled ribosome -- Dom34p (eRF1-like homologue) -- Hbs1p (eRF3-like homologue) - Promotes ribosomal subunit dissociation and peptidyl tRNAs to remove them from translation elongation stalls and recycle them - mRNA cleaved at stalled position by Dom34p which has endonuclease activity and then degraded by the exosome and/or Xrn1p No Go decay (NGD) degrades mRNA with translation elongation stalls. Dom34 appears to have endonuclease activity. Tollervey (2006) Nature 440:425

Many of the Specialized mRNA Decay Pathways Recruit Similar Proteins to the Ribosomal A Site to Trigger different mRNA Decay Mechanisms. NMD eRF eRF3 NGD DOM HSB1 NSD ? SKI7 A site binder GTPase Pathway When analyzing the components that reside in the ribosomal A site during various types of mRNA decay surveillance pathways, there is homology among the binders…eRF1 and Dom34 are similar and both bind to the A site, eRF1 during NMD and dom34 during no go decay. In addition, in all three surveillance mRNA decay pathways, there is also a GTPase protein that associates with the ribosome. eRF3 in NMD, Hsb1p in no go decay, and Ski7p in nonstop decay. Clement & Lykke-Anderson (2006) Nat Struct Mol Biol 13:299 Chen et al. (2010) Nat Struct Mol Biol 17:1233

Ribosome Extension-Mediated mRNA Decay
- Constant Spring (CS) mutation in -globin changes the UAA stop codon at the end of the mRNA into a CAA (glutamine) codon - Mutation is the most prevalent non-deletion mutation that causes -thalassemia - Ribosome continues to translate 31 codons in the 3’ UTR until a stop codon is encountered (UAA) - Level of mRNA is severely decreased due to this mutation - CS mutation causes a decrease in the mRNA half-life due to rapid induction of deadenylation - Mechanism behind this rapid deadenylation is still unknown - REMD is cell-type restricted This is a mRNA decay pathway that degrades messages where the ribosome has surpassed the stop codon and translated into the 3’ UTR and terminated translation at a stop codon located in the 3’ UTR. ….. Kong & Liebhaber (2007) NSMB 14:670

Summary of mRNA Surveillance Pathways
Summary types of mRNA surveillance pathways…. Doma & Parker (2007) Cell 131:660

3) Specialized mRNA turnover pathways - ARE-mediated mRNA decay
Now we will talk about specialized mRNA turnover pathways mediated by AREs.

ARE-Mediated mRNA Turnover
Goldstrohm & Wickens (2008) Nat Rev Mol Cell Biol 9:337 ARE - AU rich elements (50-150nts) AUUUA; UUAUUUA(U/A)(U/A); or U-rich - cis-acting element located in 3' UTRs of mRNAs - transcripts that encode proteins that require rapid changes in response to stimuli such changes in the cell cycle, growth factors, response to microorganisms, inflammatory stimuli, and environmental factors - 10% of mammalian mRNAs contain AREs - A diverse set of trans-acting proteins bind to AREs. These proteins can mediate other protein interactions that modulate mRNA stability. Various ARE-associated proteins can promote rapid mRNA turnover by promoting enhanced decapping, deadenylation, exosome recruitment, endonucleolytic cleavage or combinations of these. Alternatively, some proteins that bind to AREs can stabilize the mRNA. Now let’s talk about specialized mRNA turnover pathways that degrade specific mRNAs in response to regulatory signals. This includes ARE-mediated mRNA turnover. AREs = AU-rich elements that range from nucleotides and reside in the 3’ UTRs of mRNAs that require rapid changes in mRNA levels in response to specific stimuli. These include… A diverse set of proteins bind to AREs….they can promote turnover by…

Various Triggers Alter the Balance of Effectors
to Modulate ARE-mediated mRNA Decay The basic way an ARE regulates mRNA stability is this: 1) A trigger leads to activation of a signaling cascade. 2) Signaling cascade results in the modulation of a set of regulatory proteins by a number of ways… 3) Leads to ARE-binding proteins binding to AREs to either enhance stability= upregulation of gene expression or to enhance turnover = downregulation of gene expression Eberhardt et al. (2007) Pharm Ther 114:56

Example of ARE-Mediated Change in mRNA
Levels Before and After the DNA Damage Response Under non-damage conditions: - AUF1 competes with the PABP for poly(A) tail binding, exposing it to PARN; TTP (tristetraproline) and KSRP (KH-type splicing regulatory protein) recruit PARN and CCR4 to deadenylate prior to degradation by the exosome. Under DNA damage conditions: - Genes involved in the DNA damage response pathway are up-regulated. HuR is up-regulated and competes with AUF1 for binding to the same ARE region. Loss of AUF1 binding stabilizes PABP association with the poly(A) tail. HuR also competes with TTP and KRSP to prevent recruitment of the deadenylases and exosome. One example of ARE-mediated mRNA levels occurs in response to the DNA damage response. Cevhar & Kleiman (2010) WIRE 1:193

4) Specialized sites of cytoplasmic mRNA turnover - Processing bodies
- Exosome granules We will now discuss evidence that cytoplasmic mRNA turnover can occur at specific areas in the cytoplasm.

RNA Processing Bodies (P bodies)
- Discrete cytoplasmic granular structures that contain a reservoir of 5’ 3’ mRNA decay factors; NMD factors; RNA-induced silencing complex - Found in all eukaryotes - Size and number of P bodies depend upon the amount of RNA to be degraded Conditions that promote P body formation include:  - Glucose deprivation - Osmotic stress - UV light - Decreased translation initiation rates - Non-translating mRNA Note that all of these conditions involve moving mRNAs from a translatable pool that is ribosome-associated to a non-translatable pool that is not ribosome- associated Conditions that reduce P bodies include: - Inhibition of translation elongation (ribosome can’t dissociate from mRNA) - RNase A treatment (RNA degraded) - Increased rates of translation initiation (increase in ribosome-bound mRNA) Now let’s talk about the location of mRNA decay in RNA processing bodies or P bodies.

Kulkarni et al. (2010) Biochem Soc Trans 38: 242
Identification of Factors That Reside in P Bodies This shows the localization of P body factors. Also shown are components of P bodies. Factors Not Found in P Bodies - Translation initiation factors - Ribosomal subunits - SKI proteins & exosome Kulkarni et al. (2010) Biochem Soc Trans 38: 242

RNA Decay Intermediates Localize to P Bodies
- PolyG tract (18) in 3’ UTR blocks Xrn1p to create a mRNA decay intermediate - MS2 = bacteriophage coat protein binding site - MS2-GFP protein will bind to MS2 sites in mRNA and allows its localization by visualization of the green fluorescence protein (GFP) In this slide, we actually see mRNA intermediates localize to P bodies for decay. A reporter RNA was designed that contained a polyG tract which creates a block to Xrn1 so that the mRNA can not be completely degraded by the 5' to 3' pathway. In addition, binding sites for the MS2 protein were included in this message, that was expressed fused to GFP. This allows us to follow localization of the mRNA in the cell. The mRNA accumulated in WT yeast cells and even more so when xrn1 is deleted. The mRNA co-localized to P body markers. Sheth & Parker (2003) Science 300:805

P Bodies Disassemble After Translation Restoration
- Polysome profile (ribosomes fractionated through a sucrose gradient) - Active translation = (+) polysomes - Inactive translation = (-) polysomes - P body-associated mRNAs can return to the translatable pool The translation state is important in controlling P body formation. Conditions that lead to a departure from steady-state translation such as glucose deprivation increases the number and size of P bodies. However, this can be reversed upon the re-addition of glucose. Polysome profiles where ribosomes are segregated through a sucrose gradient were used to monitor the effiiciency of translation. The presence of polysomes indicates that translation is efficient. A loss of polysomes indicate a decrease in the translation rate. Brengues et al. (2005) Science 310:486.

Exosome Granules Are Distinct From P Bodies
and Contain ARE mRNAs and PARN exosome protein P body merge ARE mRNA PARN exosome protein merge Exosome markers are distinct from P bodies and are responsible for the degradation of ARE-containing mRNAs that are degraded by the exosome. The bodies also contain PARN. Lin (2007) JBC 282:19958

Lecture Overview 1. General mRNA turnover pathways - 5’3 & 3’5’
- Deadenylases - Decapping complex - Xrn1, exosome, DcpS 2. Aberrant RNA turnover pathways - Premature stop codons: nonsense-mediated mRNA decay (NMD) - No stop codons: non-stop mRNA decay (NSD) - Elongation stall: no-go mRNA decay (NGD) - Translation into the 3’ UTR: ribosome extension-mediated mRNA decay (REMD 3. Specialized mRNA turnover pathways - ARE-mediated mRNA turnover: AU-rich elements in the 3’ UTR are bound by proteins that modulate the stability of mRNAs in response to regulatory signals 4. Locale of mRNA turnover - P bodies: contain 5’3’ mRNA turnover machinery and degrade mRNAs that are no longer available for translation - Exosome granules: contain PARN and exosome and participate in ARE- mediated decay We discussed 4 main topics today. First we will talked about the general mRNA turnover pathways that degrade normal mRNAs that have reached their lifespan. 2) We will also discuss specialized mRNA turnover pathways that serve as a surveillance mechanism to degrade aberrant mRNAs or mRNAs that are inefficiently translated. 3) We will also discuss mRNA turnover that is induced by cellular signals that alter gene expression. 4) Finally, we discussed the location of mRNA turnover. Finally, we will discuss the site of mRNA decay with RNA granules known as processing bodies or P bodies.

2011 MECHANISMS OF CYTOPLASMIC mRNA TURNOVER IN EUKARYOTES

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2011 MECHANISMS OF CYTOPLASMIC mRNA TURNOVER IN EUKARYOTES

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Presentation on theme: "2011 MECHANISMS OF CYTOPLASMIC mRNA TURNOVER IN EUKARYOTES"— Presentation transcript:

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