8th International Meeting on Yeast Apoptosis

mRNA stability and control of cell proliferation

Cristina Mazzoni, Claudio Falcone


Most of the studies on cell proliferation examine the control of gene expression by specific transcription factors that act on transcriptional initiation. In the last few years, it became evident that mRNA stability/turnover provides an important mechanism for post-transcriptional control of gene expression. In eukaryotes, mRNAs are mainly degraded after deadenylation by decapping and exosome pathways. Mechanisms of mRNA surveillance comprise deadenylation-independent pathways such as NMD (nonsense-mediated decay), when mRNAs harbour a PTC (premature termination codon), NSD (non-stop decay, when mRNAs lack a termination codon, and NGD (no-go decay), when mRNA translation elongation stalls. Many proteins involved in these processes are conserved from bacteria to yeast and humans. Recent papers showed the involvement of proteins deputed to decapping in controlling cell proliferation, virus replication and cell death. In this paper, we will review the newest findings in this field.

  • apoptosis
  • cell proliferation
  • mRNA degradation
  • yeast

mRNA degradation

In eukaryotes, cytoplasmic mRNA decay is initiated by removal of the poly(A) tail. This first step in the turnover pathway is unique in that it is reversible and some transcripts can be readenylated and returned to polysomes. CCR4 (CC chemokine receptor 4)-NOT is the main deadenylase of the yeast Saccharomyces cerevisiae [1] formed by a large complex of nine proteins, two of which, Ccr4 and Caf1 (also known as Pop2), have exonuclease domains.

mRNAs to be destroyed can undertake one of two irreversible routes. One of these involves ‘decapping’, a process that removes the 5′ cap, followed by the degradation in the 5′–3′-direction through the Xrn1 exoribonuclease. mRNAs can also be degraded in the opposite direction by the exosome, a complex of nucleases with 3′–5′ activity. These two pathways are not mutually exclusive and their balance varies among mRNAs and organisms.

In S. cerevisiae, after the removal of the poly(A) tail, the Lsm1p–7p/Pat1p/Dhh1p complex assembles at the 3′-end of the mRNA, possibly recognizing a short oligo(A) tract. Binding of the Lsm1–7 complex may remodel the mRNA structure to allow the decapping enzyme to access the cap or alternatively, Lsm proteins may directly recruit the decapping enzymes.

The decapping enzymes Dcp1/Dcp2, together with Xrn1, form a larger complex with the Lsm1–7 proteins and co-localize at cytoplasmic foci, known as P-bodies (processing bodies), which are considered the sites of decapping and 5′–3′ mRNA decay [2,3].

Lsm complexes seem also able to inhibit RNA degradation. In fact, the loss of Lsm1–7 function results in the 3′-end trimming of deadenylated mRNAs [4] and Lsm proteins have been found implicated in stabilizing the 3′-end of intermediates in the rRNA [5] and snoRNA (small nucleolar RNA) [6] processing pathways. These effects on mRNA decay are probably mediated by the interactions of Lsm proteins with various components of the mRNA-decay machinery, including decapping enzymes (Dcp1–Dcp2), 5′–3′ exonucleases (Xrn1 and Rat1) and components of the exosome complex, which perform the 3′–5′ decay [7].

Although most transcripts undergo the standard deadenylation-dependent decay described above, there are specific mRNAs that seem to by-pass these pathways and employ several unusual routes to decay, such as the deadenylation-independent decapping [8].

Cells have evolved means for detecting and degrading aberrant transcripts for self-protection from potentially toxic protein products. While surveillance for inappropriate mRNA processing occurs in the nucleus, three pathways detect aberrant mRNP (messenger ribonucleoprotein) structures in the cytoplasm and are translation dependent [9]. The NMD (nonsense-mediated decay) detects and degrades transcripts that contain PTCs (premature termination codons) arising from frameshift mutations [10]. The NSD (non-stop decay) targets mRNAs that lack a stop codon generated by breakage or absence of an in-frame stop codon [11]. The last discovered mechanism is the NGD (no-go decay) that prevents the sequestration of translation factors to faulty transcripts by detecting stalled ribosomes on an mRNA and endonucleolytically cleaving the mRNA near the stall site. This releases the stalled ribosome and mRNA fragments, which are decayed by the exosome and Xrn1 [12].

In recent years, dysfunction of mRNA degradation, in particular in the decapping pathway, has been shown to be crucial in cell proliferation, apoptosis and viral propagation.

Role of mRNA stability in cell proliferation and apoptosis

Alterations of gene expression have an important role in tumorigenesis. This can be achieved by specific transcription factors that control transcriptional initiation; nevertheless, it is evident that mRNA turnover provides a further important mechanism for post-transcriptional control of gene expression.

The yeast Lsm1p has a critical role in decapping [13] and, although the function(s) of the human homologue hLsm1 has not been determined, there is some evidence that hLsm1p also takes part in the mRNA degradation process. In fact, components of LSm1–LSm7 complex co-localize with human Dcp1/2 and Xrn1 in discrete foci [1416] deputed to mRNA degradation [2]. Moreover, antisense-mediated reduction of hLsm1 is associated with increased p21/CIP mRNA stability [17].

Human Lsm1, also called CaSm (cancer-associated Sm-like) was originally identified because of its overexpression in pancreatic cancer, in several cancer-derived cell lines [18] as well as in metastatic tumours [19].

CaSm is also required to maintain the transformed phenotype of prostate cancer cells and it can function as an oncogene in that overexpression of this gene in NIH 3T3 and MCF10A cells leads to foci formation in vitro [17,20].

The expression of CaSm antisense, as well as the inhibition of CaSm by siRNA (small interfering RNA), has a clear effect on blocking cell proliferation of cancer cells prior to the completion of mitosis. The reduction of the CaSm protein leads to lower levels of cyclin B1 and of the CDK1 (cyclin-dependent kinase 1) that are required for the normal G2-M progression of the cell cycle, thus explaining the observed cell cycle block. Conversely, CaSm overexpression could increase CDK1 function in cancer cells, causing the by-pass of the G2 checkpoint and an increase in the proliferation rate.

Although apoptotic cells were not observed during the Ad-anti-CaSm treatment (adenoviral vector expressing antisense CaSm RNA), it could be hypothesized that apoptosis could occur in the absence of hLsm1 [17].

In fact, it has been clearly described that the reduction of cyclin B1 causes inhibition of proliferation by arresting cells in G2 phase and by inducing apoptosis in HeLa cells [21]. It can be hypothesized that cells infected with anti-CaSm-expressing virus reduce, but do not completely abolish, the hLsm1 function.

The Dhh1 (RCK/p54, Me31B) protein, a DEXD/H-box RNA helicase, is an important component of the decapping machinery that is associated with the cytoplasmic P-bodies.

Again, it has been reported that the inappropriate regulation of RCK/p54 expression is implicated in many types of tumours and in precancerous conditions, such as hepatitis C infection [2224].

In colon tumour cells, the overexpression of RCK/p54 is frequently accompanied by overexpression of c-Myc, suggesting that RCK/p54 contributes to the stabilization and increased translational efficiency of c-myc mRNA.

Considering the difficulties involved in obtaining a clear knockout of specific human genes, the use of other simpler systems, such as yeast, can be useful to unravel complex networks.

Previous studies have established yeasts as a model to study the mechanisms and the regulation of apoptosis. S. cerevisiae cells carrying a mutation in the AAA (ATPase associated with various cellular activities) gene CDC48 undergo death, showing the typical markers of apoptosis, such as DNA fragmentation, phosphatidylserine externalization and chromatin condensation [25]. VCP (valosin-containing protein), its mammalian orthologue, was subsequently linked to the regulation of apoptosis [26]. Moreover, heterologous expression of the pro-apoptotic protein hBax induces apoptosis in yeast cells and this can be counteracted by the simultaneous expression of the anti-apoptotic protein Bcl-2 [27].

In addition, exposure to low doses of H2O2 or acetic acid that are known to increase ROS (reactive oxygen species) production induces apoptosis in wild-type yeast cells indicating that, as in metazoans, ROS are a key regulator of yeast apoptosis [28,29].

In recent years, several homologues of classical apoptotic regulators have been identified and characterized, i.e. caspase (YCA1) [30], Omi [31], AIF1 [32] and EndoG (endonuclease G) [33], suggesting that the basic machinery of apoptosis is indeed present and functional also in unicellular organisms.

During the last few years, it was demonstrated that PCD (programmed cell death) in yeast, induced by internal and external triggers, can be dependent on or independent of the metacaspase gene YCA1 [34,35].

Yeast mutants in genes involved in decapping (lsm1, lsm4, lsm6, lsm7, dcp1, dcp2, dhh1 and pat1) show accumulation of mRNA degradation intermediates and an increase in intracellular ROS. These mutants undergo cell death prematurely during the stationary phase and show the typical markers of apoptosis [36,37]. A quite normal cell viability observed in one of these mutants (lsm4) was recovered following the deletion of the yeast metacaspase YCA1 gene, suggesting that caspase activity is required for apoptosis induced by increased mRNA stability [38].

Yeast mutants in genes involved in decapping also show an increase in the number of P-bodies that might represent a cellular response to the impaired mRNA degradation [3]. The number of P-bodies also increases following several apoptotic stresses and we demonstrated that this phenomenon is independent of YCA1, placing this executor of cell death downstream of the proliferation of P-bodies [39]. One possible explanation is that, following stress and apoptotic insults, the increased number of P-bodies represents a pro-survival cellular response for the sequestering of damaged or essential mRNAs that, afterwards, can be either degraded or released to translation [3941].

It is still unclear whether the defect in degradation of specific mRNAs is the basis of the apoptotic phenotypes observed in the lsm mutants.

Analysing the data concerning the negative genetic interactions of specific genes, as well those obtained with genome-wide screen (http://www.yeastgenome.org/), we found that a large group of gene mutants that are lethal/sick with lsm genes belongs to histone/chromatin modifications. Among these latter genes are hir1, hir2 and hir3 mutants.

These findings, together with the fact that the overexpression of HIR1 suppresses lsm apoptotic and premature aging phenotypes, suggest that perturbation of histone mRNA levels is crucial in the establishment of apoptosis and aging. Indeed, the Lsm1–7 proteins are involved in the degradation of histone mRNAs during cell cycle both in mammalian and yeast cells [4244].

Other interesting groups of mutants that show negative genetic interaction with lsm mutants are those involved in protein translation, DNA replication/repair, nuclear mRNA export, mitochondrial function/biogenesis and autophagy (Figure 1). Further work is necessary to clarify the role of these pathways in the apoptotic response.

Figure 1 Negative genetic interactions between Lsm1–7 mutants and other gene mutants in yeast pathways

The bacterial Hfq protein, the prokaryotic Lsm counterpart, forms a homoexameric doughnut complex and, as do the eukaryotic Lsm proteins, takes part in many aspects of RNA life. Like the Lsm complex, it can stabilize RNAs or promote their degradation.

In Escherichia coli, Hfq inactivation caused pronounced pleiotropic phenotypes, including a decrease in growth rate, biomass yield and negative supercoiling of plasmids in stationary cells, an increase in cell size, osmosensitivity, oxidation of carbon sources, sensitivity to UV light [45] and the loss of viability following heat shock [46].

Moreover, Hfq is essential for Vibrio cholerae and Pseudomonas aeruginosa virulence [47,48] and also fundamental for oxidative stress resistance and stationary phase in Salmonella enterica serotype Typhimurium [49].

It is clear that at least some phenotypes are in common with yeast lsm mutants [36,50], suggesting a strong conservation of these proteins and their role through evolution.

Role of the Lsm1p–7p/Pat1p/Dhh1p complex in viral replication

S. cerevisiae has also been used as a model in the study of virus life cycles, including virus–host interactions. To this purpose, yeast systems were developed to replicate viruses with RNA and DNA genomes that infect plants, animals and humans.

The first higher eukaryotic virus reported to replicate in yeast was BMV (Brome mosaic virus), a (+)RNA (positive-strand RNA) virus that infects plants [51].

The genome of BMV consists of three genomic RNAs with 5′ caps and tRNA-like 3′ ends. RNA1 and RNA2 encode the essential RNA replication factors 1a and 2a that direct BMV RNA replication in S. cerevisiae [51], reproducing the known features of BMV replication in plants [52].

Among the identified genes that facilitate BMV RNA replication, four of them, LSM1, LSM6, PAT1 and DHH1, belong to the complex involved in mRNA decapping [53,54].

These genes play a key role in the regulated transition of the BMV RNA from the cellular translation machinery to the site of replication [53,54]. Moreover, the viral RNA-dependent RNA polymerase co-immunoprecipitates and shows partial co-localization with Lsm1 [55].

As described above, the Lsm1p–7p/Pat1p/Dhh1p complex is involved in the movement of mRNAs out of the translating polysome pool into P-bodies, where they will be degraded or stored. This transition from a translation-competent status to a degrading-competent form, requires dramatic rearrangements in the state of the mRNA, including the loss of ribosomes and translation factors and the addition of mRNA decapping factors.

As (+)RNA genomes mimic cellular mRNAs, the Lsm1p–7p/Pat1p/Dhh1p complex could mediate rearrangements in the BMV RNA that would facilitate the loss of ribosomes and translation factors and the recognition of the 1a replicase.

Interestingly, the E. coli Hfq is required for the replication of the Q bacteriophage, a (+)RNA virus [56], indicating once more that Lsm1p–7p/Pat1p/Dhh1p has functions, conserved from prokaryotes to eukaryotes, that are exploited for their replication by some plants as well by bacteriophage (+)RNA viruses.


It is quite evident that perturbations of mRNA degradation can influence cell proliferation, cell death and viral replication in both eukaryotic and prokaryotic cells. The complex mechanisms that link the amount of translatable and untranslatable mRNAs to cell proliferation are still largely unknown and future works are needed to clarify which are the signals involved in these processes.

The unicellular yeast S. cerevisiae is a proven eukaryote model for molecular and cellular biology studies and, with the development of genomic technologies, genome-wide approaches could help in the identification of novel molecular mechanisms and their participating proteins.


  • 8th International Meeting on Yeast Apoptosis: An Independent Meeting held at Keynes College, University of Kent, Canterbury, U.K., 2–6 May 2011. Organized and Edited by Paula Ludovico (University of Minho, Braga, Portugal).

Abbreviations: BMV, Brome mosaic virus; CaSm, cancer-associated Sm-like; CDK1, cyclin-dependent kinase 1; P-body, processing body; (+)RNA, positive-strand RNA; ROS, reactive oxygen species


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