Cryptic Transcription and Early Termination in the Control of Gene Expression

Recent studies on yeast transcriptome have revealed the presence of a large set of RNA polymerase II transcripts mapping to intergenic and antisense regions or overlapping canonical genes. Most of these ncRNAs (ncRNAs) are subject to termination by the Nrd1-dependent pathway and rapid degradation by the nuclear exosome and have been dubbed cryptic unstable transcripts (CUTs). CUTs are often considered as by-products of transcriptional noise, but in an increasing number of cases they play a central role in the control of gene expression. Regulatory mechanisms involving expression of a CUT are diverse and include attenuation, transcriptional interference, and alternative transcription start site choice. This review focuses on the impact of cryptic transcription on gene expression, describes the role of the Nrd1-complex as the main actor in preventing nonfunctional and potentially harmful transcription, and details a few systems where expression of a CUT has an essential regulatory function. We also summarize the most recent studies concerning other types of ncRNAs and their possible role in regulation

Crystal structures of the Gon7/Pcc1 and Bud32/Cgi121 complexes provide a model for the complete yeast KEOPS complex.

International audienceThe yeast KEOPS protein complex comprising Kae1, Bud32, Cgi121, Pcc1 and Gon7 is responsible for the essential tRNA threonylcarbamoyladenosine (t(6)A) modification. Deletion of genes coding for the KEOPS subunits also affects telomere elongation and transcriptional regulation. In the present work, the crystal structure of Bud32/Cgi121 in complex with ADP revealed that ADP is bound in the catalytic site of Bud32 in a canonical manner characteristic of Protein Kinase A (PKA) family proteins. We found that Gon7 forms a stable heterodimer with Pcc1 and report the crystal structure of the Pcc1-Gon7 heterodimer. Gon7 interacts with the same Pcc1 region engaged in the archaeal Pcc1 homodimer. We further show that yeast KEOPS, unlike its archaeal counterpart, exists as a heteropentamer in which Gon7, Pcc1, Kae1, Bud32 and Cgi121 also adopt a linear arrangement. We constructed a model of yeast KEOPS that provides structural insight into the role of Gon7. The model also revealed the presence of a highly positively charged crater surrounding the entrance of Kae1 that likely binds tRNA

Neutralizing Aptamers from Whole-Cell SELEX Inhibit the RET Receptor Tyrosine Kinase

Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacological challenge. Specific oligonucleotide ligands (aptamers) can be generated for a variety of targets through the iterative evolution of a random pool of sequences (SELEX). Nuclease-resistant aptamers that recognize the human receptor tyrosine kinase RET were obtained using RET-expressing cells as targets in a modified SELEX procedure. Remarkably, one of these aptamers blocked RET-dependent intracellular signaling pathways by interfering with receptor dimerization when the latter was induced by the physiological ligand or by an activating mutation. This strategy is generally applicable to transmembrane receptors and opens the way to targeting other members of this class of proteins that are of major biomedical importance

Architecture of the yeast Pol III pre-termination complex and pausing mechanism on poly(dT) termination signals

RNA polymerase (Pol) III is specialized to transcribe short, abundant RNAs, for which it terminates transcription on polythymine (dT) stretches on the non-template (NT) strand. When Pol III reaches the termination signal, it pauses and forms the pre-termination complex (PTC). Here, we report cryoelectron microscopy (cryo-EM) structures of the yeast Pol III PTC and complementary functional states at resolutions of 2.7-3.9 Å. Pol III recognizes the poly(dT) termination signal with subunit C128 that forms a hydrogen-bond network with the NT strand and, thereby, induces pausing. Mutating key interacting residues interferes with transcription termination in vitro, impairs yeast growth, and causes global termination defects in vivo, confirming our structural results. Additional cryo-EM analysis reveals that C53-C37, a Pol III subcomplex and key termination factor, participates indirectly in Pol III termination. We propose a mechanistic model of Pol III transcription termination and rationalize why Pol III, unlike Pol I and Pol II, terminates on poly(dT) signals

Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration

mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5ʹ exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3ʹ was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favouring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.This work has been supported by grants from the Ministerio de Economía, Industria y Competitividad – Agencia Estatal de Investigación, and European Union funds (FEDER) [BFU2016-77728-C3-1-P to S. C.],[BFU2016-77728-C3-3-P to J.E.P-O & P.A], [BFU2016-77728-C3-2-P to F.N.] and RED2018‐102467‐T to J.E.P-O, F.N. and S.C.; by FPI grants from the Spanish Government to V.B and A.C-B; by the Regional Andalusian Government [BIO271 and US-1256285 to S. C.], [BIO258 and FEDER-UJA 1260360 to F.N.] and from the Regional Valencian Government [AICO/2019/088 to J.E.P-O]. Funding for open access charge: [BFU2016-77728-C3-1-P]

Gcn4 misregulation reveals a direct role for the evolutionary conserved EKC/KEOPS in the t6A modification of tRNAs

The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific profile that is highly enriched in targets of the Gcn4p transcriptional activator. GCN4 expression was found to be activated at the translational level in mutants via the defective recognition of the inhibitory upstream ORFs (uORFs) present in its leader. We show that EKC/KEOPS mutants are defective for the N6-threonylcarbamoyl adenosine modification at position 37 (t6A37) of tRNAs decoding ANN codons, which affects initiation at the inhibitory uORFs and provokes Gcn4 de-repression. Structural modeling reveals similarities between Kae1 and bacterial enzymes involved in carbamoylation reactions analogous to t6A37 formation, supporting a direct role for the EKC in tRNA modification. These findings are further supported by strong genetic interactions of EKC mutants with a translation initiation factor and with threonine biosynthesis genes. Overall, our data provide a novel twist to understanding the primary function of the EKC/KEOPS and its impact on several essential cellular functions like transcription and telomere homeostasis