Which of the following best describes a mechanism involved in differential gene expression

Site-Specific Antitermination: Attenuators and Riboswitches

Upstream intrinsic termination is used to regulate expression of many operons through mechanisms that alternatively permit or prevent transcription termination in a leader sequence or initially translated region (Figure 3). Alternative RNA structures (e.g., a Riboswitch or Ribosensor) in the nascent transcript dictate whether transcription continues or is terminated based on the formation or lack of formation of a terminator structure in the RNA. The RNA fold that forms is influenced in most examples by small molecules (i.e., metabolites, cations, and tRNAs) that directly bind to the RNA and affect folding. Both positive and negative transcription-based riboswitches have been described. Classical riboswitches rely on interactions with small molecules to dictate RNA structure, although examples of protein-influenced riboswitches are known.

As a term, attenuation is most often reserved for translation-coupled regulation of intrinsic termination, whereby positioning of a trailing ribosome influences formation of a downstream terminator. The classical example of such regulation is found in the trp operon, where stalling of the ribosome at tryptophan codons, due to limited availability of charged aminoacyl-tRNATrp, determines which sequences of the transcript can hybridize, and thus whether a terminator hairpin can be formed. Examples of riboswitches and attenuators have been described for Eukaryotes. However, although the prokaryotic Archaea appear poised to take advantage of these systems, there is no evidence for such regulation.

Termination

Transcription termination occurs in a reaction coupled to RNA 3′-end processing. Most eukaryotic mRNA precursors are cleaved in a site-specific manner in the 3′-untranslated region, followed by polyadenylation of the upstream cleavage product. A large number of proteins are involved in these reactions. The exact mechanism of coupling between 3′-end processing and transcription termination remains unclear. Termination is accompanied by dephosphorylation of the Pol II CTD, but the precise timing of Pol II dephosphorylation is also unclear. Dephosphorylation is required for the reinitiation of transcription, because Pol II can only join an initiation complex in its unphosphorylated form. The CTD phosphatase Fcp1 plays a key role in Pol II dephosphorylation and recycling. Fcp1 binds to Pol II via Rpb4. Rpb4 apparently recruits Fcp1 to the vicinity of the CTD, because the Rpb4/7 complex binds near the linker that connects the Pol II core to the CTD.

Transcription Cycle: Termination

Transcription termination is the final step of gene expression, which plays an important role in making an end of RNA without affecting unnecessary gene expression from downstream genes. This is particularly important for the ‘gene rich’ genome of bacteria. There are two types of termination: ρ-dependent termination and ρ-independent intrinsic termination. The ρ-dependent termination requires a hexameric helicase protein ρ, which loads onto nascent RNA strands at rut (rho utilization) sites (Figure 1) followed by stimulating an RNA-dependent adenosine triphosphatase (ATPase) activity. Coupling of ATP hydrolysis translocates ρ in the 5′→ 3′ direction along the RNA toward the EC. Eventually, ρ bumps into the RNAP and pulls out RNA from the EC, causing the release of the transcript and dissociation of RNAP from DNA.

The intrinsic terminators contain short self-complementary GC-rich sequences followed by an oligo-A tract on the template DNA sequence. Transcription of such sequences is accompanied by pausing of RNAP due to backtracking, which ensures the formation of a hairpin-like RNA structure. The RNA hairpin destroys the nucleic acid contacts with RNAP, which leads to collapse of the transcription bubble and disintegration of the EC.

Abstract

Transcription termination in eukaryotic cells involves the recognition of polyadenylation signals (PAS) that signal the site of pre-mRNA cleavage and polyadenylation. Most eukaryotic genes contain multiple PAS that are used by alternative polyadenylation (APA), a co-transcriptional process that increases transcriptomic diversity and modulates the fate of the mRNA and protein produced. However, current tools to pinpoint the relationship between mRNAs in different subcellular fractions and the gene expression outcome are lacking, particularly in primary human immune cells, which, due to their nature, are challenging to study. Here, we describe an integrative approach using subcellular fractionation and RNA isolation, chromatin-bound and nucleoplasmic RNA-Sequencing, 3′ RNA-Sequencing and bioinformatics, to identify accurate APA mRNA isoforms and to quantify gene expression in primary human macrophages. Our protocol includes macrophage differentiation and polarization, co-culture with cancer cells, and gene silencing by siRNA. This method allows the simultaneous identification of macrophage APA mRNA isoforms integrated with the characterization of nuclear APA events, the identification of the molecular mechanisms involved, as well as the gene expression alterations caused by the cancer-macrophage crosstalk. With this methodology we identified macrophage APA mRNA signatures driven by the cancer cells that alter the macrophage inflammatory and transcriptomic profiles, with consequences for macrophage physiology and tumor evasion.

3.4.3 Articulavirales

Transcription termination by the influenza polymerase occurs at stretches of uracil residues encoded near the 5′ ends of the genome segments, resulting in a poly-A stuttering mechanism like the polymerases of the mononegaviruses (Fig. 1C) [280,281]. A cryo-EM snapshot of the terminating influenza polymerase reveals a disordered template RNA, indicative of the stuttering mechanism and slippage movement of the polyU template region within the active site to facilitate polyadenylation [59]. Polymerase stuttering continues until the added polyA tail reaches approximately 150 bases in length [174,282]. The polyadenylated viral mRNA dissociates from the transcriptase complex and is delivered to ribosomes in the cytosol via the host mRNA export machinery. The 5′ hook remains bound in the active site through polyadenylation and product dissociation [59]. Interestingly, polymerase stuttering appears to be controlled, in part, by the limiting length of RNA connecting the 5′ hook and the polyU tract in the active site. As more template becomes extruded through the exit channel, the length of RNA connecting the 5′ hook and the template engaged in the RdRP active site becomes shorter. Eventually, the polymerase can no longer translocate the anchored template RNA through the active site, which may facilitate stuttering at the polyU sequence and explains why the 5′ terminus is not copied during mRNA synthesis [59]. The importance of polymerase engagement with terminal 5′ sequences for efficient polyadenylation was recognized over two decades ago from extensive RNA mutagenesis and in vitro polyadenylation experiments [173,174,176]. Recent structural efforts have shed light on the importance of these earlier findings [59]. Additional regulators of influenza polyadenylation and transcription termination, such as the potential role of host mRNA processing factors [283], remain under investigation.

While the 5′ end of the template RNA must remain in its binding pocket throughout polyadenylation and mRNA termination, these sequences must be copied during vRNA and cRNA replication to ensure synthesis of correct 3′ promoter regions. Precisely how this structured 5′ hook is removed from its binding pocket remains unknown. It is possible that short (~ 22–27 nt) 5’ RNAs expressed during influenza virus infection could compete in trans to displace the 5′ end and allow for complete terminal copying [284–286]. This would also release the physical constraint of the tethered 5′ hook connected to the RNA being copied, possibly eliminating polymerase stuttering at the upstream polyU sequence. Thus, the influenza virus polymerase could terminate replication by a runoff mechanism at the precise 5′ end of the template RNA. However, the precise mechanisms of switching between mRNA termination via polyA stuttering and v/cRNA termination remain unknown.

D YaeO–Rho: Inhibition of Rho-Dependent Transcription Termination

Transcription termination is the process where a nascent RNA is released from its complex with RNA polymerase and the DNA template. In bacteria, two main mechanisms of transcription termination have been described. These mechanisms, commonly referred to as Rho-independent and Rho-dependent termination, are essential for the regulation of bacterial gene expression (Richardson and Greenblatt, 1996). Rho-dependent termination requires the presence of a hexameric helicase, Rho (Brown et al., 1981), an essential transcription factor that binds nucleic acids at specific termination sites (rut), and translocates along the RNA until it reaches the transcription complex (Geiselmann et al., 1993; Platt, 1994; Richardson, 1996). One of the Rho-specific inhibitors of transcription is the product of the yaeO gene, which reduces termination in the Rho-dependent bacteriophage terminator tL1, and upstream the autogenously regulated gene rho (Pichoff et al., 1998). Overexpression of YaeO can cause the pleiotropic suppression of conditional lethal mutations in cell division and heat shock genes, such as ftsQ, ftsA, grpE, groEL, and groES (Pichoff et al., 1998).

We first determined the NMR solution structure of YaeO that revealed a topologically similar fold to that of the RNA-binding domain of small ribonucleoproteins (Sm-fold) (Gutierrez et al., 2007). In order to understand the mechanism of transcription termination inhibition by YaeO, NMR experiments were used to characterize the interaction of YaeO with Rho in vitro. We used the N-terminal fragment (residues 1–130), referred as Rho130, that corresponds to the primary RNA binding site of Rho and has been shown to be a good model of Rho-oligonucleotide interactions (Briercheck et al., 1998). The titration resulted in mapping the binding site of Rho130 on YaeO, which consists of the N- and C-termini, helix α1, and strands β3, β4, β5, and β7. These regions localize to one edge of the β-sandwich with clustered acidic residues. As the structure of Rho130 has been also determined (Briercheck et al., 1998), we mapped the YaeO binding site on Rho130 that partially overlaps with the RNA binding surface, suggesting a mechanism of transcription termination inhibition.

As NMR titration data for the YaeO–Rho interaction was obtained for both proteins, a docking model of the complex was calculated using HADDOCK (Dominguez et al., 2003). AIRs were derived from the NMR titration data by selecting residues with both the biggest chemical shifts and solvent accessibility. The resulting model is compatible with the hexameric Rho structure and reflects the charge complementarities of the interacting protein surfaces (Fig. 6B). This is consistent with in vitro binding results that show that the YaeO–Rho interaction is salt dependent and can be disrupted at high ionic strengths (Pichoff et al., 1998). Importantly, the structural model was used to design the D14K, E19K, and E52K YaeO mutants that prevent inhibition of Rho activity using an in vivo β-galactosidase assay (Gutierrez et al., 2007).

4.2.1 Polymerase II

Although transcription termination has been much less explored than its initiation, the past 25 years has revealed that the well timed and accurate release of the nascent transcript and RNA polymerases from the DNA template is crucial for the fate of the RNA as well as for overall genome maintenance.

Two mechanisms have been proposed for this process in the case of mRNA coding genes, allosteric (antiterminator) and torpedo (Fig. 7.2A; reviewed in Refs. [67–69]). The allosteric model postulates that 3′ processing factors trigger conformational changes in the transcription elongation complex, which facilitates the dissociation of antiterminator factors and/or binding of termination factors [70]. The torpedo model was first proposed by Connelly and Manley [71] and Proudfoot [72], who suggested that the 3′ pre-mRNA product generated by the cleavage and polyadenylation apparatus is attacked by 5′-3′ exoribonuclease activity, which degrades the Pol II-associated RNA faster than it is synthesized, while removing the polymerase from the template. In fact, both termination pathways are often orchestrated and, with some exceptions, can be unified in one allosteric-torpedo model [73].

Later work revealed that Rat1 in yeast and Xrn2 in humans were responsible for the torpedo activity [27,74,75] and that lack of either enzyme, as well as the Rat1 activator Rai1 in yeast, leads to a termination defect, which manifests as an extensive transcription read-through. It is also possible that a plant Rat1 homologue, Arabidopsis AtXRN3, acts as a transcription termination factor similarly to Rat1/Xrn2. High-throughput approaches revealed an accumulation of noncoding transcripts from the 3′-end of mRNA and microRNA (miRNA) genes in the xrn3 knockdown mutant, which may correspond to transcriptional read-through molecules [76]. Thus, AtXRN3 may participate in a global RNA 3′-end surveillance as a result of its torpedo termination activity.

The torpedo mechanism depends on the exonuclease catalytic activity of Rat1, and not merely on its presence [27]. The monophosphorylated RNA substrate for Rat1/Xrn2 during mRNA synthesis is generated as a result of a cotranscriptional cleavage (CoTC) at the polyadenylation site by the mRNA 3′-end formation machinery. More precisely, this process is performed by the Ysh2 component of the CF II in yeast or the corresponding CPSF-73 subunit of the human cleavage and polyadenylation specificity factor (CPSF) (reviewed in Ref. [77]). Two factors were proposed to contribute to efficient termination: the strength of the poly(A) signal and the action of the exonuclease degrading the 5′ cleavage product, which facilitates pausing of the polymerase downstream of the poly(A) site and promotes its release [75]. On the other hand, impaired Rat1-dependent termination does not abolish proper poly(A)-site recognition and mRNA cleavage [27].

Additional Rat1/Xrn2 entry sites may arise through autocatalytic CoTC downstream of the poly(A) site in mammals [74] or endonucleolytic cleavage by Rnt1 in yeast [78]. The latter case was reported as a fail-safe termination mechanism for protein-coding genes, which, together with the pathway mediated by the Nrd1/Nab3/Sen1 complex (NRD), provides a backup mode for Pol II release.

Although Rat1/Xrn2 is required for efficient Pol II termination, Rat1 fails to trigger termination in vitro and its 5′-3′ degradation activity is not sufficient to promote polymerase dissociation in vivo[73,79]. Consistently, yeast Xrn1 targeted to the nucleus is capable of the cotranscriptional degradation of nascent RNA but does not rescue termination defects caused by a Rat1 deficiency. These observations suggest that the exonuclease catching up with the polymerase is necessary but not sufficient to destabilize the elongation complex, and that an additional element of the torpedo mechanism operates during termination. It has been postulated that a Rat1 unique feature, the extended tower domain, not present in Xrn1 proteins, may be responsible for its termination properties [25]. It is conceivable that the tower domain undergoes certain modifications or serves as an interface for interactions with termination-enhancing factors. One of the possible candidates for factors stimulating Rat1-dependent termination is the RNA helicase Sen1, which is a key component of the termination pathway for noncoding RNAs in yeast (see below) that also contributes to the termination of protein-coding genes, with the strongest effect on shorter mRNAs [80–82]. Sen1 interacts via its N-terminal domain with the largest Pol II subunit, Rpb1 [83] and impairing Sen1 helicase activity impairs genome-wide Pol II distribution over coding and noncoding genes [81]. The human Sen1 homologue, Senataxin, was shown to directly promote Xrn2-mediated transcription termination by resolving the RNA:DNA hybrids (R-loops) formed behind the elongating Pol II, predominantly at G-rich pause sites downstream of the poly(A) site [84]. This activity facilitates the access of Xrn2 to poly(A) site 3′ cleavage products and thus contributes to the recruitment of the torpedo exonuclease. The mode of action of yeast Sen1 is thought to be similar [85].

An unanswered question is how Rat1 is recruited to the elongating polymerase. Several observations support Rat1 association via proteins interacting with the Pol II C-terminal domain (CTD) through their CTD-interacting domain (CID), namely the Pcf11 subunit of the yeast cleavage factor IA (CFIA) and Rtt103 (regulator of Ty1 transposition 103). Rat1 and Rai1 are present at the promoters and coding regions with a strong enrichment at the 3′-ends of genes. The functional interaction between Rat1 and Pcf11 was shown to facilitate the mutual corecruitment of both factors: Rat1 links termination to the 3′-end formation by stimulating the recruitment of 3′-end processing factors, especially the CFIA subunits Pcf11 and Rna15, while Pcf11 contributes to Rat1 association over the poly(A) site [73]. Also, human Pcf11 was shown to enhance degradation of nascent RNA and promote transcription termination [86]. In turn, Rtt103, which also associates near the 3′-ends of genes, copurifies with Rat1, Rai1, and Pcf11, and together with Pcf11 cooperatively recognizes the Ser2-phosphorylated CTD of the elongating Pol II [87,88]. On the other hand, Rat1 distribution over genes is not altered in the absence of Rtt103 [87]. In addition, Rat1 also terminates transcription by Pol I [29,82], which lacks a CTD, suggesting that Rat1 recruitment may also occur via other mechanisms. Indeed, the association of hXrn2 was reported to be mediated by p54nrb/PFS (protein-associated splicing factor), which are multifunctional proteins involved in transcription, splicing, and polyadenylation [89]. The role of p54nrb/PFS in Pol II transcription termination was also demonstrated in C. elegans[90].

In addition to mRNAs, Pol II synthesizes a broad range of noncoding transcripts, including small nuclear RNAs and snoRNAs and a variety of unstable RNAs. In yeast, the transcription of these relatively short species is terminated mainly by an alternative NRD complex, composed of the RNA-binding proteins Nrd1 and Nab3 and the ATP-dependent helicase Sen1 [80,81,91–93]. Since this mechanism most likely does not entail a cotranscriptional endonucleolytic cleavage [92], it is the Sen1 RNA:DNA hybrid-unwinding activity that is thought to be essential for polymerase dissociation, perhaps in a manner similar to bacterial Rho DNA–RNA helicase. Rho probably destabilizes the elongation complex by translocating along and removing the nascent RNA from the polymerase [94]. An alternative, allosteric model posits that Rho loads onto polymerase and induces rearrangements in the enzyme active site at termination sites [95]. Both modes of action may well apply for Sen1 helicase.

Although Rat1 is recruited to snoRNA genes, particularly intronic ones, NRD-dependent termination of snoRNA transcripts is not impaired when it is missing, and it was, therefore, suggested that it participates in some premature termination events [92,96]. It is still possible, however, that the Rat1 torpedo mechanism may contribute to transcription termination of snoRNAs, such as U3 or snR40, whose 3′-end is released by Rnt1 cleavage that exposes the remaining nascent transcript 5′-end for Rat1 attack [97,98].

Surprisingly, Rat1 was shown to colocalize with Pcf11 at telomeres and at the Pol III-transcribed tRNA, 5S rRNA, and SCR1 genes [96]. The functional significance of these observations is currently unclear, but alternative termination modes of these RNAs, and the degradation of aberrant transcripts or the processing of unstable noncoding RNAs, such as telomeric TERRA [99], are a possibility (Table 7.1).

Table 7.1. Yeast Rat1-cooperating proteins

Predicted transcription attenuation in bacteria

Gene regulation by transcription termination–antitermination, often called transcription attenuation, is a strategy commonly used by bacteria to sense a specific metabolic signal and enables a response that directs RNA polymerase to either terminate transcription or transcribe the downstream genes of an operon (for a review of these mechanisms see ‘Posttranscriptional Regulation’).

The decision whether to terminate transcription is often based on the selective arrangement of one of the two mutually exclusive RNA secondary structures in the nascent transcript, the antiterminator and the terminator. Transcription attenuation web page compiles a computer-based predict transcription attenuators for fully sequence genomes. The computer predictions are based on the search of potential alternative RNA-hairpin structures in the leader sequence that precedes a particular gene or operon. The predicted transcription attenuators in this database are clustered by organisms or by COG classification.

6.21.2.3 mRNA Stability and Processing

While regulation by transcription termination and translation initiation predominates in the riboswitch systems that have been characterized to date, there are a few examples that operate at other steps of gene expression. The glmS riboswitch is unique in that binding of the effector promotes transcript self-cleavage by activation of a ribozyme activity inherent in the glmS RNA.5 The cleaved transcript is then subject to degradation by RNase J1,6 resulting in decreased expression of the downstream gene (Figure 3). In systems of this type, the synthesis rate of the full-length transcript is constant. However, the steady-state level of the transcript changes in response to effector binding. Riboswitches have also been identified in plants and fungi and they have been shown to affect transcript stability and mRNA splicing; these all belong to the Thi box family, and respond to thiamine pyrophosphate (TPP). The eukaryotic riboswitches identified to date are located in the 3′-untranslated region (3′-UTR) of the transcript or are positioned within an intron.7,8

3.8 Optimization and troubleshooting