The role of RNA Polymerase II-dependent transcription elongation in the cross-talk between mRNA synthesis and decay.

The role of RNA Polymerase II-dependent transcription elongation in the cross-talk between mRNA synthesis and decay Victoria Begley Doctoral thesis 2018 INDEX 1. Introduction. ......................................................................................................................... 1 Gene expression. ........................................................................................................................... 3 Transcription. ................................................................................................................................ 3 1. Initiation: ....................................................................................................................... 5 2. Elongation: .................................................................................................................... 6 3. Termination: ................................................................................................................ 10 mRNA degradation. ..................................................................................................................... 11 The main deadenylator: the Ccr4-Not complex. ................................................................. 13 The RNA exonuclease Xrn1. ................................................................................................ 14 From transcription to decay: mRNA imprinting. ......................................................................... 15 From mRNA decay to transcription. ............................................................................................ 16 Biological significance of the transcription/decay cross-talk. ..................................................... 18 2. Objectives. .......................................................................................................................... 21 3. Results. ................................................................................................................................ 25 Chapter 1: Molecular characterization of mutants potentially affecting the transcription/mRNA decay crosstalk. ........................................................................................ 27 Mutants show slight variation in gene expression that is exacerbated by longer gene length. ................................................................................................................................. 31 The selected mutants show altered Pol II occupancy and active Pol II. ............................. 34 Total mRNA concentrations are not dependent on any single variable studied. ............... 43 Growth rate correlates with transcription and degradation. ............................................. 45 Cell volume negatively correlates with transcription and degradation. ............................ 46 Pol II speed is important for the cross-talk mechanism. .................................................... 49 Degradation rates and synthesis rates correlate. ............................................................... 51 Materials and methods. ...................................................................................................... 55 Chapter 2: Mathematical Modelling and computational simulation of the cross-talk between mRNA transcription and decay. ................................................................................................. 61 How to model such a complex system (ODD protocol). ..................................................... 65 Definition of the system and its agents. ............................................................................. 65 Programming the system. ................................................................................................... 68 Model variants. ................................................................................................................... 71 First results. ......................................................................................................................... 73 Calibrating the model.......................................................................................................... 73 Final results. ........................................................................................................................ 75 Materials and methods. ...................................................................................................... 79 Chapter 3: Genome-wide analysis of the contribution of Xrn1 to transcription elongation using CRAC. ................................................................................................................................. 81 The deletion of XRN1 led to a reduction in transcription rates. ......................................... 84 RNAs labelled with 4-thiouracil cannot be efficiently degraded by the exosome in an xrn1∆ mutant. ..................................................................................................................... 94 There is a global increase in arrested Pol II when Xrn1 is rapidly depleted from the cell. . 97 Forcing Xrn1 out of the nucleus led to changes in early Pol II elongation but not in late elongation. ........................................................................................................................ 105 Materials and methods. .................................................................................................... 112 4. Discussion. ........................................................................................................................ 119 The characterization of transcription by various different methods enables a better understanding of the transcriptional defects present in each mutant. .................................... 121 mRNA degradation is not only determined by function. .......................................................... 123 Correlations between mRNA turnover with growth rate and cell volume validate our results. ................................................................................................................................................... 124 Pol II elongation rate can influence mRNA degradation. .......................................................... 125 mRNA degradation can influence transcription elongation. .................................................... 126 Metagene analysis showed defects in transcription elongation in the absence of Xrn1. ........ 127 The controversy of the role of Xrn1 in the cross-talk between mRNA decay and transcription. ................................................................................................................................................... 128 All results confirm a strong cross-talk between transcription and mRNA decay. .................... 130 Working model. ......................................................................................................................... 131 The Cross-talk beyond transcription and mRNA decay............................................................. 135 5. Conclusions. ...................................................................................................................... 137 6. Bibliography. ..................................................................................................................... 141 1 1. Introduction. 2 3 Gene expression. Gene expression is the process by which information contained in the DNA is converted into a functional gene product that dictates cell function. These products are often proteins, but in non-protein coding genes the product is a functional RNA. The most important molecule in gene expression is RNA, whose main job is to transfer the genetic code from the nucleus, where the DNA is, to the cytoplasm, where it can be translated into protein by ribosomes. The RNA levels in a cell are a result of the balance between its synthesis (transcription) and its degradation. Thus, these processes play a critical role in determining what proteins are present in a cell and in what amounts. Therefore, to understand how gene expression works, we need to understand how these two processes work. Transcription. Conventionally, transcription is regarded as one of the most important steps in the RNA life cycle since it is responsible for the synthesis of the transcript itself. In addition, by processing the 5’ and 3’ ends of the pre-mRNA, the transcription machinery prepares it for correct export, translation and degradation in the cytoplasm (Svetlov and Nudler, 2013). The substrate of transcription is not naked DNA but chromatin. The nucleosome is the basic unit of DNA coiling in eukaryotic chromosomes that compacts the genome in the form of chromatin. It is composed of 145-147 base pairs of DNA wrapped around a histone octamer containing two copies of histones H2A, H2B, H3 and H4 (Luger et al., 1997). Chromatin contains a repeating array of nucleosomes that are spaced every 200 ± 40 base pairs throughout the genome. Following nucleosome disruption by multiprotein complexes, the transcription machinery can interact with the uncoiled DNA and begin the transcription process (Kornberg, 2001). Transcription of the eukaryotic genome is carried out by the nuclear RNA polymerase I (Pol I), Pol II and Pol III. Whereas Pol I transcribes the ribosomal RNA genes (rRNA); Pol II transcribes messenger RNA (mRNA) and a subset of non-coding RNAs (ncRNA); and Pol III transcribes transfer RNAs (tRNAs), 5S rRNA and the bulk of ncRNAs (Sainsbury et al., 2015). These three polymerases present a similar structure and share a 10 subunit core with specific subunits on the periphery (Cramer et al., 2008). Pol II transcription is the first step in gene expression, therefore we will focus on the study of this polymerase. Yeast Pol II is a multiprotein complex of 514 kDa consisting of twelve subunits (Cramer, 2004). These proteins are coded by the genes RPB1-12 and the two specific subunits of this polymerase are the heterodimer Rpb4/7. All RPB genes are essential for cell viability except for 4 RPB4 and RPB9 (Woychik and Young, 1989; Woychik et al., 1991). The crystal structure of transcribing Pol II (Gnatt et al., 2001) revealed that Rpb1 and Rpb2 form a centre cleft, with the smaller subunits arrayed around the periphery. In this cleft is the active site, marked by an Mg2+ ion, where the biogenesis of nascent RNA takes place. Comparison with a higher resolution crystal structure later revealed that the polymerase is divided into four mobile modules, including a clamp that swings over the active centre (Cramer et al., 2014). It also revealed another metal ion at the active site that could possibly be exchangeable during RNA synthesis. Duplex DNA is thought to enter the main cleft or “jaw” and unwind before the active site. RNA synthesis then takes place, leading to a 9bp DNA-RNA hybrid that is held in place by the clamp. After passing through the active site, the DNA path is blocked by a protein “wall” forcing it to pass up the wall at nearly right angles to the incoming DNA in the cleft. The nascent RNA and the transcribed DNA abandons the polymerase through an exit channel. A pore in the floor of the cleft beneath the active site allows entry of substrate nucleoside triphosphates (NTPs) and also allows exit of RNA during retrograde movement of the polymerase on the DNA. Figure 1. Side view of the RNA polymerase II transcribing complex (Klug, 2001). In light grey the front of the enzyme is shown, and in dark grey the back part. The duplex DNA, in blue and green, can be seen entering the polymerase through the “jaw”. The template strand coding for RNA is in blue, the non-template strand is in green. The RNA in the DNA-RNA hybrid in the active centre region is in red. The 3' (growing) end of the RNA is located adjacent to an active site Mg2+ ion (pink sphere). Opposite to the entering site of DNA, a protein “wall” forces the DNA-RNA to make a nearly right angle turn to exit the enzyme. The NTPs enter through a funnel-shaped opening on the underside of the enzyme and gain access to the active centre through a pore. One distinct feature of Pol II is the presence of a C-terminal extension on its largest subunit (Rpb1), referred to as the C-terminal domain (CTD). It consists of tandem repeats of the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, repeated 26 times in yeast. Each amino acid in the CTD can be post-translationally modified. The CTD is highly conserved and essential for viability in all organisms (Corden, 2013). During transcription, the CTD is 5 phosphorylated at specific amino acids depending on the progression along the gene. These phosphorylations serve as a signature of the different Pol II transcription steps, and they create binding sites for chromatin regulators and RNA processing factors that are required in specific stages of transcription (Suh et al., 2016). The combination of posttranslational modifications in the CTD is known as the CTD code (Buratowski, 2003). The transcription process by Pol II can be divided into three steps: initiation, elongation and termination. 1. Initiation: For initiation, Pol II assembles with the general transcription factors (GTFs) TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH at promoter DNA to form the pre-initiation complex (PIC) (Grünberg and Hahn, 2013). GTFs cooperate with Pol II to bind to and open promoter DNA, to initiate RNA synthesis and stimulate the escape of Pol II from the promoter (Sainsbury et al., 2015). The PIC assembles in nucleosome-free promoter regions that are flanked by an upstream −1 nucleosome and a downstream +1 nucleosome (Venters and Pugh, 2010). In yeast, the transcription start site (TSS) is located in the upstream border of the +1 nucleosome, suggesting that this nucleosome potentially regulates access to the TSS (Albert et al., 2007). During PIC assembly, a dephosphorylated Pol II is recruited to promoters through interactions with TFIIB and the Mediator Complex (Myers et al., 1998). This complex serves as a central scaffold within the PIC, and the different mediator subunits make contact with various transcription factors, including activators, co-activators, GTFs, and subunits of Pol II (Ansari and Morse, 2013). The first DNA contact of the GTFs occur at the TATA or TATA-like gene promoters. Both promoters are located at a fixed, short distance from the TSS that is recognized by the TATA-binding protein (TBP) (Venters and Pugh, 2010). However, on the one hand, the TATA promoter is made up of a consensus sequence (TATA(A/T)A(A/T)(A/G)), and on the other hand, the TATA-like promoters possess a sequence with two or less mismatches to the TATA box consensus (Sung Rhee and Franklin Pugh, 2012). Approximately 80-90% of all Saccharomyces cerevisiae genes are TATA-like, and in this case the TBP was thought to be a part of the TFIID complex which guided it to gene promoters. For the rest of genes (10-20%) the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex was thought to direct TBP to TATA box containing Pol II promoters (Bhaumik and Green, 2002). Thus, the expression of most mRNAs was considered to be dominated by either the transcription factor TFIID or SAGA. However, recently this notion has been challenged. Two publications show that both transcription factors are necessary for nearly all yeast genes, and the differences observed before are just 6 differential dependence on SAGA or TFIID (Baptista et al., 2017; Warfield et al., 2017). Genome-wide analysis showed that TATA-containing promoters are highly regulated and generally stress responsive, with expression levels being extremely high or low. In contrast, TATA-like promoters are those genes referred to as housekeeping genes and are more constitutively active with less regulation requirements (Hahn and Young, 2011). The first step in canonical PIC assembly is the sequence-specific binding of TBP to promoter DNA, inducing a bend. Then, the GTFs TFIIA and TFIIB stabilize the TBP-DNA complex by flanking TBP on both sides. Subsequently, Pol II associated to TFIIF joins the resulting complex and finally, TFIIE and TFIIH bind and complete the PIC. Pol II cannot melt DNA itself, although it can maintain an open transcription bubble. Thus, TFIIE and TFIIH are required to melt the DNA in the presence of ATP (figure 2). Once the template DNA is opened and melted, RNA synthesis begins (Sainsbury et al., 2015). In early transcription, abortive initiation occurs with frequency, resulting in short unstable transcripts. When the transcript reaches 10 nucleotides, promoter escape is favoured over abortive initiation and the complex stabilizes. However, a transcript length of a minimum of 25 nucleotides is necessary to achieve productive initiation and accordingly, transcription elongation can commence (Saunders et al., 2006). Figure 2. Scheme of the PIC assembly and initiation of transcription. The initiation competent Pol IITFIIF complex joins the initial PIC formed by TFIID, who contains TBP, TFIIA and B. TBP recognizes the promoter of the gene and induces a bend in the DNA. Next, TFIIE and TFIIH bind to complete the PIC and melt DNA forming the transcription bubble. Once this occurs, transcription can begin. 2. Elongation: The Kin28 subunit of TFIIH mediates the transition from transcription initiation to elongation through phosphorylation of serines in the 5th position of the CTD (Valay et al., 1995). When approximately 25 nucleotides of nascent RNA have been synthesized, the cap structure is attached to its 5′ end. The capping enzyme (guanylyltransferase) specifically recognizes the phosphorylated Ser-5 CTD repeats, which then catalyzes the addition of a methylguanosine cap to the 5′ end of nascent mRNA, which is a signal for productive transcription initiation 13 mRNA to decay that is carried out by Xrn1, a 5’-3’ exoribonuclease. This is the major degradation pathway that occurs in the cytoplasm. Decapping and the 5’-3’ degradation are coupled, as Xrn1 nuclease interacts directly with Pat1. Some mRNAs can be degradaded without the removal of the poly(A) tail. These mRNAs are aberrant and are subjected to the NMD surveillance system. Xrn1 degrades these aberrant mRNAs following 5’ cap removal by Dcp2 initiated by the NMD factors (Conti and Izaurralde, 2005). Deletions of the main enzymes of either of the degradation pathways do not result in the total accumulation of aberrant mRNA, which suggests that these enzymes work in cooperation (Siwaszek et al., 2014). The main deadenylator: the Ccr4-Not complex. The Ccr4-Not complex is essential and present in all eukaryotes. Structurally, it consists of 9 core subunits in yeast: Ccr4, Caf1, Caf40, Caf130, and Not1-5 (Collart and Panasenko, 2012). This core complex is between 0.9-1.2 MDa in size. It has been described, however, that the complex can also contain a combination of Dhh1, Dbf2, Caf4, Caf16, and Btt1 forming a larger complex of >1.9 MDa (Miller and Reese, 2012). In 2001, the core complex was purified by the Not1 subunit and viewed by electron microscopy (Nasertorabi et al., 2011). This allowed visualization of the global shape of Ccr4-Not that revealed an L-shaped complex with two arms of similar length (figure 6). Ccr4, an endonuclease–exonuclease–phophatase (EEP), is the main deadenylase in the complex. Ccr4 is incorporated into the whole complex through Caf1 via a leucine-rich repeat motif (LRR) (Siwaszek et al., 2014). Caf1 possess an exonuclease motif (RNase D) and its deletion shows defects in deadenylation in vivo, similar to that of ccr4∆ (Tucker et al., 2001). However, it is dispensable for catalysis in vitro (Tucker et al., 2002). In this context, two different roles have been proposed for the Caf1 protein: first, it may be responsible for deadenylation under some circumstances given the conservation of some of the key residues in the active site. And second, it may enhance the function of Ccr4 by stabilizing the complex Figure 6. Cartoon representation of the L-shape of the core Ccr4- Not complex defined by electron microscopy (Nasertorabi et al., 2011) with the expected position of the core subunits on the Not1 scaffold (Collart, 2016). 14 or providing additional interactions with the mRNA. This was proposed because in the absence of Caf1 a defect in deadenylation was found, however, strains containing a full-length but catalytically inactive Caf1 showed no phenotype (Parker and Song, 2004). The Pan2-Pan3 complex is also involved in deadenylation in yeast and it seems that the activity of both deadenylating complexes is partially redundant. Deletion of ccr4 gives slower deadenylation rates, but only the ccr4∆ pan2∆ double mutant accumulates poly(A) tailed mRNAs and has severe growth defects (Tucker et al., 2002). Not1 is the scaffold of the complex and in S. cerevisiae it is the only subunit essential for viability (Maillet et al., 2000). It is the largest subunit of the complex and does not possess enzymatic activity. Caf130 and Caf40, which are also incorporated into the yeast complex, possess no clear function. Not2, Not3, Not4 and Not5 form a Not module interacting with the C-terminus of Not1 (Bhaskar et al., 2013). Not2, Not3 and Not5 are closely associated, and together they create a platform that may contribute to the functions of the Ccr4-Not complex, including deadenylation. Finally, Not4 is an active E3 ubiquitin ligase. It is involved in protein turnover regulation and has been found to have a specific role in protein quality control that extends beyond regulation of deadenylation (Halter et al., 2014). The RNA exonuclease Xrn1. Yeast Xrn1 is a large, nonessential, highly conserved cytoplasmic 5’-3’ exoribonuclease whose well defined biochemical function is to degrade mRNA containing a free 5’ monophosphate end (Nagarajan et al., 2013a). Xrn1 degrades a wide range of cytoplasmic RNAs, including NMD-substrates, noncoding RNAs and a novel class of long non-coding RNAs (lncRNAs) called XRN1-sensitive Unstable-Transcripts (XUTs) (Wery et al., 2016). Deletion of Xrn1 leads to stabilization of mRNAs on a global scale, consistent with its role in the major cytoplasmic mRNA decay pathway (Haimovich et al., 2013a; Sun et al., 2013; Wery et al., 2016). Xrn1 possesses an essential paralog that degrades RNAs in the nucleus named Rat1. Xrn1 and Rat1 are highly conserved in their N-terminal nuclease domain and are functionally interchangeable: yeast Rat1 can substitute for the function of Xrn1 when localized to the cytoplasm, while Xrn1 tagged with a nuclear localization signal (NLS) can rescue the lethal phenotype of a temperature-sensitive Rat1 mutant, rat1-1ts (Johnson, 1997). However, both Xrn1 and Rat1 are involved in processing of nuclear structural RNA (snoRNA and pre-rRNA) (Petfalski et al., 1998). This suggests that Xrn1 may be able to locate to the nuclear or nucleolar compartment. 15 From transcription to decay: mRNA imprinting. Two yeast Pol II proteins, Rpb4 and Rpb7, separate from the core and form a heterodimer that shuttles between the nucleus, where it functions in transcription, and the cytoplasm (Selitrennik et al., 2006), where it functions in the major mRNA decay pathway (Lotan et al., 2005, 2007). Rpb4/7 binds Pol II transcripts cotranscriptionally and is associated with the mRNA throughout its life (Goler-Baron et al., 2008), modulating its translation (Harel-Sharvit et al., 2010) and degradation. Efficient translation in the cytoplasm depends on association of Rpb4/7 with Pol II in the nucleus, therefore, Pol II can be considered a key regulator of the major stages of gene expression and acts as a ‘‘mRNA coordinator” (Haimovich et al., 2013b). Another identified “mRNA coordinator” is Pab1 that, as mentioned previously, is involved in the 3′-end processing of mRNA. It then regulates the export of bound mRNA (Brune et al., 2005), translation initiation (Kahvejian et al., 2005), translation termination (Hosoda et al., 2003), deadenylation and decapping (Wiederhold and Passmore, 2010). Impairing the import of Pab1 into the nucleus delays mRNA export out of the nucleus (Brune et al., 2005). Thus, the nuclear and cytoplasmic roles of Pab1 are thought to be coupled. The yeast Ccr4-Not complex has also been found to function both in transcription and degradation (figure 7). The complex was initially identified as a transcription factor (Collart and Struhl, 1994; Draper et al., 1994). Later, it became apparent that the complex possessed many other functions such as nuclear RNA processing (Azzouz et al., 2009), mRNA export (Kerr et al., 2011), translation quality control and protein degradation (Collart and Panasenko, 2012) and, most importantly as one of the two major deadenylases (Tucker et al., 2002). Ccr4-Not mutants display many phenotypes observed in the rpb4 deleted strain, therefore, suspicions were formed as to whether the complex could also coordinate transcription and degradation. In a recent paper, a connection was made between Rpb4/7 and Ccr4-Not (Babbarwal et al., 2014). They suggest that Rpb4/7 is required for Ccr4-Not to bind elongation complexes and rescue arrested Pol II, thus heightening these suspicions. Decisively, an imprinting mechanism through the Ccr4-Not complex was recently discovered (Gupta et al., 2016). In this model Not1 imprinting plays an important role in coupling different steps of gene expression, facilitating both translation of the imprinted mRNA, specifically ribosomal protein and mitochondrial genes, and its subsequent decay. This Not1 imprinting is both dependent on Not5 and transcription elongation, with more imprinting occurring in the presence of Not5 and with higher backtracking levels of Pol II. Therefore, they concluded that the complex is indeed linked to mRNA coordination, from transcription to mRNA decay. 16 Figure 7. The many functions of the Ccr4-Not complex connecting transcription, mRNA degradation and translation. 1. Ccr4-Not has been found to be involved in transcription initiation. 2. It is involved in transcription elongation. 3. It has been described to be involved in RNA processing and mRNA imprinting. 4. In mRNA export through the nuclear pore complex (NPC). 5. It is involved in translation associated to translating ribosomes. 6. In mRNA degradation by deadenylation. 7. In protein quality control. 8. And in the translation of Pol II. From mRNA decay to transcription. The Ccr4-Not complex has also been linked to coordinate mRNA decay to its transcription (figure 7). Once the complex has finished its role in deadenylation, it can shuttle into the nucleus where it has been implicated in various aspects of transcription. On one hand there is evidence indicating it is involved in transcription initiation. First of all, Ccr4-Not subunits are present at promoters (Venters et al., 2011) and tethering of Ccr4-Not complex subunits to promoters can activate or repress transcription (Benson et al., 1998). Second, Not4 can regulate the recruitment of certain transcription factors to promoters (Gulshan et al., 2012). 17 Finally, subunits of the Ccr4-Not complex show both genetic and physical interactions with TFIID and SAGA subunits (Miller and Reese, 2012). Gene expression analyses of ccr4-not mutants indicate that SAGA-dependent genes are mostly affected (Cui et al., 2008), and recently, it was shown that Not5 is important for the functional integrity of SAGA (Kassem et al., 2017). On the other hand, it has also been found to be involved in transcription elongation. First, the deletion of Ccr4-Not subunits show synthetic lethal interactions with the deletion of other elongation factors: Paf1c, Spt16, TFIIS and Spt5 (Denis et al., 2001). In addition, some Ccr4-Not mutants are sensitive to the elongation inhibitors 6-azauracil (6-AU) and mycophenolic acid. Second, specific transcription elongation assays show defects in several Ccr4-Not complex mutants (Gaillard et al., 2009; Kruk et al., 2011). Third, the complex was found to cross-link to the open reading frames (ORFs) of genes (Venters et al., 2011). Its recruitment to genes is likely due to its ability to interact directly with the Rpb4/7 module of Pol II (Babbarwal et al., 2014). Finally, Ccr4-Not was found to rescue backtracked polymerases (Dutta et al., 2015; Kruk et al., 2011), as mentioned previously. This might explain the synthetic lethal interaction with TFIIS, another factor involved in rescuing backtracked Pol II. Furthemore, the Ccr4-Not complex has been shown to be connected to the nuclear RNA degradation machinery with an impact on mRNA processing (Azzouz et al., 2009). It can also interact with nuclear ribonucleoproteins and the nuclear pore complex, suggesting a role in mRNA export (Kerr et al., 2011). Some of these factors may be the target of the Not4 ubiquitin ligase as ubiquitination has been implicated in the control of mRNA processing and export (Gwizdek et al., 2006). In the cytoplasm, the Ccr4-Not complex possesses a role in translation. This role is supported by findings such as reduced levels of total polysomes in yeast cells lacking Not2, Not4 or Not5. Also, newly produced proteins have been found to aggregate massively in not2, not4, and not5 mutants (Panasenko and Collart, 2012). This role in translation is particularly important in the case of Not5 as it has been shown to contribute to the synthesis of Rpb1 (Villanyi et al., 2014). This connects translation to transcription. Xrn1 is another interesting component of the degradation machinery for the study of crosstalk as it has been recently linked to transcription (Haimovich et al., 2013a; Sun et al., 2013). Haimovich et al. attributed a stimulatory role of Xrn1 in transcription. They show that Xrn1 was present upstream of the transcription start sites of the genes most affected by the loss of Xrn1. In contrast, the research by Sun et al. described a negative effect of Xrn1 on global 18 transcription rates. Despite this controversy, both studies concluded that Xrn1 plays a crucial role in maintaining mRNA homeostasis in response to alterations in either synthesis or mRNA decay. The implication of Xrn1 in cross-talk might be more intense in some gene categories, such as highly transcribed genes, mainly those encoding ribosome biogenesis and translation factors (Medina et al., 2014a). Biological significance of the transcription/decay cross-talk. Cross-talk between transcription and degradation of mRNAs is vital to coordinate hundreds to thousands of transcripts simultaneously, providing a rapid gene expression response during diverse cellular processes and stresses. Some of these processes are cell growth and cell division, cell cycle, response to stress or other environmental cues (Das et al., 2017; Haimovich et al., 2013b). During exponential growth, cell volume increases over time until it undergoes cell division. During this time, mRNA levels are at risk of becoming diluted. Consequently, transcription and degradation must be regulated in order to maintain mRNA homeostasis since under or overexpression of factors could be deleterious to living cells. Supporting this idea, a correlation was found between both transcription and mRNA degradation with growth rates (García-Martínez et al., 2016a), and also with cell volume (Mena et al., 2017). It was proposed that a change in growth rate between generations could be damaging to cells if they are not accompanied by an adjustment in transcription and degradation rates (Chávez et al., 2016). For instance, if daughter cells inherit exceedingly stable mRNAs from their mothers, correct phenotypic response to stimuli could be inhibited. This correct adjustment becomes of great importance when it comes to faster proliferating cells. More than 10% of the protein-coding genes are cell cycle regulated in S. cerevisiae (Spellman et al., 1998). These genes alternate between high and low expression periodically. It has been proposed that, at least for some of these genes, cross-talk between transcription an decay aids in the timely expression of genes necessary to enter into the different cell cycle phases (Trcek et al., 2011). Additionally, cross-talk is postulated to shape the expression of distinct gene clusters which undergo cell-cycle-dependent regulation, such as genes involved in DNA synthesis, cytokinesis, budding and other cell cycle-specific events (Wittenberg and Reed, 2005). Various stresses, such as osmotic stress, heat shock or DNA damage require a rapid response by increasing the expression of some genes and repressing the expression of others. In these 19 cases, it is essential to reach the newly required steady state mRNA levels and maintain them. This sharp rise or decrease in gene expression can be achieved more efficiently thanks to a coordination between synthesis and decay (Romero-santacreu et al., 2009). Furthermore, this cross-talk provides an efficient way to maintain global mRNA concentrations within certain limits (±50%) in different physiological situations in S. cerevisiae (Benet et al., 2017; Sun et al., 2013). Even during stress, transcription and mRNA decay was shown to correlate with growth rate, thus cross-talk remains even in non-steady state conditions (García-Martínez et al., 2016b). In a recent paper (Dori-Bachash et al., 2011), the evolution of transcription and degradation rates in two closely related yeast species was assessed. Remarkably, they found that around half of the evolutionary differences in mRNA degradation are coupled to evolutionary differences in transcription. These findings gave rise to the idea that coupling transcription with decay facilitates evolvability as it reduces the number of mutations required to evolve a new trait, such as a new regulatory mechanism (Haimovich et al., 2013b). Transcription and mRNA degradation also appears to be functionally coupled in mammalian cells. In mice and humans, differences in mRNA degradation were found to compensate transcriptional differences. Additionally, this compensation found to be evolutionary conserved between the two species (Dori-Bachash et al., 2012). Furthermore, several families of mammalian proteins have been associated to cross-talk: BTG/Tob, TIS11, and KSRP. For example, in the first protein family, BTG/tob, the BTG domain is implicated in the interaction with components of transcription as well as mRNA decay such as CNOT7 and CNOT8 which are part of the Ccr4-Not complex. BTG/Tob are involved in the control of cell cycle progression in a variety of cell types and their loss of expression contributes to carcinogenesis and tumour growth (Winkler, 2010). Thus, cross-talk may be important for human diseases. Another link of cross-talk between transcription and mRNA decay to human diseases is the exploit of the feedback mechanism by a gamma herpes virus. This virus encodes a endonuclease, SOX, that cleaves most cellular mRNAs. SOX generates many new fragments of RNA that require Xrn1 for their degradation. Consequently, Xrn1 is retained in the cytoplasm and cannot perform its function in transcription, leading to transcriptional repression in the nucleus. Indeed the virus uses this feedback mechanism to facilitate viral gene expression, as most of its genes escape this transcriptional repression, and infect its host (Abernathy et al., 2015). Finally, the pathogenic fungus Cryptococcus neoformans was also found to use crosstalk in its infecting process. In this case, the fungus uses the coupling between transcription 20 and degradation to adapt to the temperature of its host and to promote pathogenicity. 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