High-resolution analysis of cell-state transitions in yeast suggests widespread transcriptional tuning by alternative starts

Background: The start and end sites of messenger RNAs (TSSs and TESs) are highly regulated, often in a cell-type-specific manner. Yet the contribution of transcript diversity in regulating gene expression remains largely elusive. We perform an integrative analysis of multiple highly synchronized cell-fate transitions and quantitative genomic techniques in Saccharomyces cerevisiae to identify regulatory functions associated with transcribing alternative isoforms. Results: Cell-fate transitions feature widespread elevated expression of alternative TSS and, to a lesser degree, TES usage. These dynamically regulated alternative TSSs are located mostly upstream of canonical TSSs, but also within gene bodies possibly encoding for protein isoforms. Increased upstream alternative TSS usage is linked to various effects on canonical TSS levels, which range from co-activation to repression. We identified two key features linked to these outcomes: an interplay between alternative and canonical promoter strengths, and distance between alternative and canonical TSSs. These two regulatory properties give a plausible explanation of how locally transcribed alternative TSSs control gene transcription. Additionally, we find that specific chromatin modifiers Set2, Set3, and FACT play an important role in mediating gene repression via alternative TSSs, further supporting that the act of upstream transcription drives the local changes in gene transcription. Conclusions: The integrative analysis of multiple cell-fate transitions suggests the presence of a regulatory control system of alternative TSSs that is important for dynamic tuning of gene expression. Our work provides a framework for understanding how TSS heterogeneity governs eukaryotic gene expression, particularly during cell-fate changes

Chromatin-sensitive cryptic promoters putatively drive expression of alternative protein isoforms in yeast

Cryptic transcription is widespread and generates a heterogeneous group of RNA molecules of unknown function. To improve our understanding of cryptic transcription, we investigated their transcription start site (TSS) usage, chromatin organization, and posttranscriptional consequences in Saccharomyces cerevisiae We show that TSSs of chromatin-sensitive internal cryptic transcripts retain comparable features of canonical TSSs in terms of DNA sequence, directionality, and chromatin accessibility. We define the 5' and 3' boundaries of cryptic transcripts and show that, contrary to RNA degradation-sensitive ones, they often overlap with the end of the gene, thereby using the canonical polyadenylation site, and associate to polyribosomes. We show that chromatin-sensitive cryptic transcripts can be recognized by ribosomes and may produce truncated polypeptides from downstream, in-frame start codons. Finally, we confirm the presence of the predicted polypeptides by reanalyzing N-terminal proteomic data sets. Our work suggests that a fraction of chromatin-sensitive internal cryptic promoters initiates the transcription of alternative truncated mRNA isoforms. The expression of these chromatin-sensitive isoforms is conserved from yeast to human, expanding the functional consequences of cryptic transcription and proteome complexity

A functional connection between translation elongation and protein folding at the ribosome exit tunnel in Saccharomyces cerevisiae

Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5′ region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel

A complete set of nascent transcription rates for yeast genes

The amount of mRNA in a cell is the result of two opposite reactions: transcription and mRNA degradation. These reactions are governed by kinetics laws, and the most regulated step for many genes is the transcription rate. The transcription rate, which is assumed to be exercised mainly at the RNA polymerase recruitment level, can be calculated using the RNA polymerase densities determined either by run-on or immunoprecipitation using specific antibodies. The yeast Saccharomyces cerevisiae is the ideal model organism to generate a complete set of nascent transcription rates that will prove useful for many gene regulation studies. By combining genomic data from both the GRO (Genomic Run-on) and the RNA pol ChIP-on-chip methods we generated a new, more accurate nascent transcription rate dataset. By comparing this dataset with the indirect ones obtained from the mRNA stabilities and mRNA amount datasets, we are able to obtain biological information about posttranscriptional regulation processes and a genomic snapshot of the location of the active transcriptional machinery. We have obtained nascent transcription rates for 4,670 yeast genes. The median RNA polymerase II density in the genes is 0.078 molecules/kb, which corresponds to an average of 0.096 molecules/gene. Most genes have transcription rates of between 2 and 30 mRNAs/hour and less than 1% of yeast genes have >1 RNA polymerase molecule/gene. Histone and ribosomal protein genes are the highest transcribed groups of genes and other than these exceptions the transcription of genes is an infrequent phenomenon in a yeast cell

The Rocketbox Library and the Utility of Freely Available Rigged Avatars

As part of the open sourcing of the Microsoft Rocketbox avatar library for research and academic purposes, here we discuss the importance of rigged avatars for the Virtual and Augmented Reality (VR, AR) research community. Avatars, virtual representations of humans, are widely used in VR applications. Furthermore many research areas ranging from crowd simulation to neuroscience, psychology, or sociology have used avatars to investigate new theories or to demonstrate how they influence human performance and interactions. We divide this paper in two main parts: the first one gives an overview of the different methods available to create and animate avatars. We cover the current main alternatives for face and body animation as well introduce upcoming capture methods. The second part presents the scientific evidence of the utility of using rigged avatars for embodiment but also for applications such as crowd simulation and entertainment. All in all this paper attempts to convey why rigged avatars will be key to the future of VR and its wide adoption

Comparing and Evaluating Real Time Character Engines for Virtual Environments

As animated characters increasingly become vital parts of virtual environments, then the engines that drive these characters increasingly become vital parts of virtual environment software. This paper gives an overview of the state of the art in character engines, and proposes a taxonomy of the features that are commonly found in them. This taxonomy can be used as a tool for comparison and evaluation of different engines. In order to demonstrate this we use it to compare three engines. The first is Cal3D, the most commonly used open source engine. We also introduce two engines created by the authors, Piavca and HALCA. The paper ends with a brief discussion of some other popular engines

The 3-Base Periodicity and Codon Usage of Coding Sequences Are Correlated with Gene Expression at the Level of Transcription Elongation

Background: Gene transcription is regulated by DNA transcriptional regulatory elements, promoters and enhancers that are located outside the coding regions. Here, we examine the characteristic 3-base periodicity of the coding sequences and analyse its correlation with the genome-wide transcriptional profile of yeast. Principal Findings: The analysis of coding sequences by a new class of indices proposed here identified two different sources of 3-base periodicity: the codon frequency and the codon sequence. In exponentially growing yeast cells, the codon-frequency component of periodicity accounts for 71.9 % of the variability of the cellular mRNA by a strong association with the density of elongating mRNA polymerase II complexes. The mRNA abundance explains most of the correlation between the codon-frequency component of periodicity and protein levels. Furthermore, pyrimidine-ending codons of the four-fold degenerate small amino acids alanine, glycine and valine are associated with genes with double the transcription rate of those associated with purine-ending codons. Conclusions: We demonstrate that the 3-base periodicity of coding sequences is higher than expected by the codon usage frequency (CUF) and that its components, associated with codon bias and amino acid composition, are correlated with gene expression, principally at the level of transcription elongation. This indicates a role of codon sequences in maximising the transcription efficiency in exponentially growing yeast cells. Moreover, the results contrast with the common Darwinia

An mRNA decapping mutant deficient in P body assembly limits mRNA stabilization in response to osmotic stress

Yeast is exposed to changing environmental conditions and must adapt its genetic program to provide a homeostatic intracellular environment. An important stress for yeast in the wild is high osmolarity. A key response to this stress is increased mRNA stability primarily by the inhibition of deadenylation. We previously demonstrated that mutations in decapping activators (edc3∆ lsm4∆C), which result in defects in P body assembly, can destabilize mRNA under unstressed conditions. We wished to examine whether mRNA would be destabilized in the edc3∆ lsm4∆C mutant as compared to the wild-type in response to osmotic stress, when P bodies are intense and numerous. Our results show that the edc3∆ lsm4∆C mutant limits the mRNA stability in response to osmotic stress, while the magnitude of stabilization was similar as compared to the wild-type. The reduced mRNA stability in the edc3∆ lsm4∆C mutant was correlated with a shorter PGK1 poly(A) tail. Similarly, the MFA2 mRNA was more rapidly deadenylated as well as significantly stabilized in the ccr4∆ deadenylation mutant in the edc3∆ lsm4∆C background. These results suggest a role for these decapping factors in stabilizing mRNA and may implicate P bodies as sites of reduced mRNA degradation

Towards the Composition of Services by End-Users: A Mobile-Based Solution

[EN] Nowadays, we live surrounded by heterogeneous and distributed services that are available to people anytime and anywhere. Even though these services can be used individually, it is through their synchronized and combined usage that end-users are provided with added value. However, existing solutions to service composition are not targeted at ordinary end-users. In fact, these solutions require technical knowledge to deal with the technological heterogeneity in which they are offered to the market. To this end, the paper presents a tool-supported platform that is aided by: (1) EUCalipTool, an end-user mobile tool that implements a Domain Specific Visual Language, which has been specifically designed to compose services on mobile devices; (2) a Faceted Service Registry, which plays the role of gateway between service implementations and end-users, hiding technological issues from the latter when including services in a composition; and (3) a Generation Module, which transforms end-user descriptions into BPMN specification that are interpreted by an execution infrastructure developed for that purpose.This work has been developed with the financial support of the Spanish State Research Agency under the project TIN2017-84094-R and co-financed with ERDF.Valderas, P.; Torres Bosch, MV.; Pelechano Ferragud, V. (2020). Towards the Composition of Services by End-Users: A Mobile-Based Solution. Business & Information Systems Engineering. 62(4):305-321. https://doi.org/10.1007/s12599-019-00617-zS305321624Amir R, Zeid A (2004) A UML profile for service-oriented architectures. In: Companion to the 19th annual ACM SIGPLAN conference on object-oriented programming systems, languages, and applications, Vancouver. ACM, New York, pp 192–193Athreya B, Bahmani F, Diede A, Scaffidi C (2012) End-user programmers on the loose: a study of programming on the phone for the phone. In: IEEE symposium on visual languages and human-centric computing, Innsbruck. IEEE, pp 75–82Atooma (2015) Atooma, a touch of magic. https://www.atooma.com/ . Accessed 1 Oct 2018Ayora C, Torres V, Weber B, Reichert M, Pelechano V (2013) Enhancing modeling and change support for process families through change patterns. In: Nurcan S et al (eds) Enterprise, Business-Process and Information Systems Modeling. BPMDS 2013, EMMSAD 2013, vol 147. Lecture Notes in Business Information Processing. Berlin, Heidelberg, pp 246–260Benedek J, Miner T (2002) Measuring desirability: new methods for evaluating desirability in a usability lab setting. In: Proceedings from the Usability’s Professionals Association (UPA)Broke J (1996) SUS. A “quick and dirty” usability scale. In: Jordan P et al (eds) Usability evaluation in industry. Taylor & Francis, London, pp 189–194Cuccurullo S, Francese R, Risi M, Tortora G (2011) MicroApps development on mobile phones. In: Costabile MF, Dittrich Y, Fischer G, Piccinno A (eds) End-User Development. IS-EUD 2011, vol 6654. Lecture Notes in Computer Science. Berlin, Heidelberg, pp 289–294Danado J, Paternò F (2014) Puzzle: a mobile application development environment using a jigsaw metaphor. J Vis Lang Comput 25(4):297–315Danado J, Davies M, Ricca P, Fensel A (2010) An authoring tool for user generated mobile services. In: Berre AJ, Gómez-Pérez A, Tutschku K, Fensel D (eds) Future internet—FIS 2010. FIS 2010, vol 6369. Lecture Notes in Computer Science. Berlin, Heidelberg, pp 118–127Dey AK, Sohn T, Streng S, Kodama J (2006) iCAP: interactive prototyping of context-aware applications. In: Fishkin KP, Schiele B, Nixon P, Quigley A (eds) Pervasive Computing. Pervasive 2006, vol 3968. Lecture Notes in Computer Science. Berlin, Heidelberg, pp 254–271Engeström Y, Miettinen R, Punamäki RL (1999) Perspectives on activity theory. Cambridge University Press, CambridgeErmagan V, Krüger IH (2007) A UML2 profile for service modeling. In: Engels G, Opdyke B, Schmidt DC, Weil F (eds) Model Driven Engineering Languages and Systems. MODELS 2007, vol 4735. Lecture Notes in Computer Science. Berlin, Heidelberg, pp 360–374Galitz WO (2002) The essential guide to user interface design: an introduction to GUI. Design principles and techniques. Wiley, New YorkGuillen J, Miranda J, Berrocal J, Garcia-Alonso J, Murillo JM, Canal C (2014) People as a service: a mobile-centric model for providing collective sociological profiles. IEEE Softw 31(2):48–53Häkkilä J, Korpipää P, Ronkainen S, Tuomela U (2005) Interaction and end-user programming with a context-aware mobile application. In: Costabile MF, Paternò F (eds) Human-Computer Interaction—INTERACT 2005, Lecture Notes in Computer Science, vol 3585. Berlin, Heidelberg, pp 927–937IFTTT (2015) IFTTT, if this then that. https://IFTTT.com/ . Accessed 1 Oct 2018Klusch M, Sycara K (2001) Brokering and matchmaking for coordination of agent societies: a survey. In: Omicini A, Zambonelli F, Klusch M, Tolksdorf R (eds) Coordination of Internet Agents. Springer, Berlin, Heidelberg, pp 197–224Locale (2015). http://www.twofortyfouram.com . Accessed 1 Oct 2018Lucci G, Paternò F (2014) Understanding end-user development of context-dependent applications in smartphones. In: Sauer S, Bogdan C, Forbrig P, Bernhaupt R, Winckler M (eds) Human-Centered Software Engineering. HCSE 2014, Lecture Notes in Computer Science, vol 8742. Berlin, Heidelberg, pp 182–198Paolucci M, Kawamura T, Payne TR, Sycara K (2002) Semantic matching of web services capabilities. In: Horrocks I, Hendler J (eds) The Semantic Web—ISWC 2002, Lecture Notes in Computer Science, vol 2342. Berlin, Heidelberg, pp 333–347Renger M, Kolfschoten GL, de Vreede GJ (2008) Challenges in collaborative modeling: a literature review. In: Advances in enterprise engineering I, vol 10, Montpellier, pp 61–77Repenning A, Ioannidou A (2006) What makes end-user development tick? 13 design guidelines. In: End user development. Human-computer interaction series, vol 9. Springer, Berlin, pp 51–85Rumbaugh J, Jacobson I, Booch G (2004) The unified modeling language reference manual. Pearson, LondonSegal J (2005) Two principles of end-user software engineering research. ACM SIGSOFT Softw Eng Notes 30(4):1–5Serral E, Valderas P, Pelechano V (2013) Context-adaptive coordination of pervasive services by interpreting models during runtime. Comput J 56(1):87–114Tasker (2015) Tasker, total automation for Android. http://tasker.dinglisch.net/ . Accessed 1 Oct 2018Uden L, Valderas P, Pastor O (2008) An activity-theory-based model to analyse web application requirements. Inf Res 13(2):1Valderas P, Pelechano V, Pastor O (2006) A transformational approach to produce web application prototypes from a web requirements model. Int J Web Eng Technol 3(1):4–42Valderas P, Torres V, Mansanet I, Pelechano V (2017) A mobile-based solution for supporting end-users in the composition of services. Multimed Tools Appl 76(15):16315–16345Workflow.is (2018) Workflow. Spend less taps, get more done. https://workflow.is/ . Accessed 1 Oct 2018Yu J, Sheng QZ, Han J, Wu Y, Liu C (2012) A semantically enhanced service repository for user-centric service discovery and management. Data Knowl Eng 72:202–21

Mutually exclusive sense–antisense transcription at FLC facilitates environmentally induced gene repression

Antisense transcription through genic regions is pervasive in most genomes; however, its functional significance is still unclear. We are studying the role of antisense transcripts (COOLAIR) in the cold-induced, epigenetic silencing of Arabidopsis FLOWERING LOCUS C (FLC), a regulator of the transition to reproduction. Here we use single-molecule RNA FISH to address the mechanistic relationship of FLC and COOLAIR transcription at the cellular level. We demonstrate that while sense and antisense transcripts can co-occur in the same cell they are mutually exclusive at individual loci. Cold strongly upregulates COOLAIR transcription in an increased number of cells and through the mutually exclusive relationship facilitates shutdown of sense FLC transcription in cis. COOLAIR transcripts form dense clouds at each locus, acting to influence FLC transcription through changed H3K36me3 dynamics. These results may have general implications for other loci showing both sense and antisense transcription