Real time monitoring of endogenous cytoplasmic mRNA using linear antisense 2′-O-methyl RNA probes in living cells

Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2′-O-methyl RNA probes, which have different interactive fluorophores at the 5′-end of one probe and at the 3′-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells

Role of the RNA-Binding Protein Nrd1 in Stress Granule Formation and Its Implication in the Stress Response in Fission Yeast

We have previously identified the RNA recognition motif (RRM)-type RNA-binding protein Nrd1 as an important regulator of the posttranscriptional expression of myosin in fission yeast. Pmk1 MAPK-dependent phosphorylation negatively regulates the RNA-binding activity of Nrd1. Here, we report the role of Nrd1 in stress-induced RNA granules. Nrd1 can localize to poly(A)-binding protein (Pabp)-positive RNA granules in response to various stress stimuli, including heat shock, arsenite treatment, and oxidative stress. Interestingly, compared with the unphosphorylatable Nrd1, Nrd1DD (phosphorylation-mimic version of Nrd1) translocates more quickly from the cytoplasm to the stress granules in response to various stimuli; this suggests that the phosphorylation of Nrd1 by MAPK enhances its localization to stress-induced cytoplasmic granules. Nrd1 binds to Cpc2 (fission yeast RACK) in a phosphorylation-dependent manner and deletion of Cpc2 affects the formation of Nrd1-positive granules upon arsenite treatment. Moreover, the depletion of Nrd1 leads to a delay in Pabp-positive RNA granule formation, and overexpression of Nrd1 results in an increased size and number of Pabp-positive granules. Interestingly, Nrd1 deletion induced resistance to sustained stresses and enhanced sensitivity to transient stresses. In conclusion, our results indicate that Nrd1 plays a role in stress-induced granule formation, which affects stress resistance in fission yeast

About drawing: theoretich and visual study

Esta dissertação tem como objeto de estudo o desenho, entendendo-o como um fenômeno complexo que se situa na zona de tensão entre arte e técnica, fazer e pensar. É possível considerá-lo como meio, processo e fim. Os objetivos são mapear o significado da palavra desenho, analisar o fenômeno e também os procedimentos envolvidos na ação de desenhar. O trabalho foi dividido em três capítulos, sendo o primeiro voltado para as definições do termo estudado, a etimologia e estudos do desenho como processo; o segundo capítulo aborda algumas especificidades do desenho e desafios de elaborar uma pesquisa sobre desenho; e o terceiro capítulo reúne diversas entrevistas feitas com artistas e/ou teóricos especialistas no assunto. Consideramos esse estudo como teórico-visual, pois foram utilizados recursos gráficos como ferramenta de pesquisa, em conjunto com o material escrito. Exemplos: infografia, ilustração, diagramas, narrativas gráficas, entre outros. A abordagem da pesquisa é no âmbito da estética descrito por Pareyson, de modo que seja apenas especulativa e não normativa. Ou seja, buscando definir conceitos e não normas.The subject of this thesis is the drawing (desenho) as a complex phenomenon situated in the tension area between art and technique, doing and thinking. It can be considered as means, process and goal. The objectives are to map the meaning of the word \"drawing\", analyze the phenomenon and also the procedures involved in the action of drawing. The work has been divided into three chapters: The first focuses on the definitions of the studied term, the etymology and the study of drawing as a process; the second chapter discusses some of the drawing specificities and the challenges of developing a research about it; and the last one gathers several interviews with artists and/or theoreticians that are experts in the field. We consider this study as both theoretical and visual, since as graphical features (computer graphics, illustration, diagrams, graphic narratives, among others) have been used as a research tool, combined with bibliography. The research approach situates itself in the context of aesthetics defined by Pareyson, so that it is only speculative and not normative. In other words, we defined concepts and not rules

Fission yeast Prp4p kinase regulates pre-mRNA splicing by phosphorylating a non-SR-splicing factor

We provide evidence that Prp4p kinase activity is required for pre-mRNA splicing in vivo and show that loss of activity impairs G(1)–S and G(2)–M progression in the cell cycle. Prp4p interacts genetically with the non-SR (serine/arginine) splicing factors Prp1p and Prp5p. Bacterially produced Prp1p is phosphorylated by Prp4p in vitro. Prp4p and Prp1p also interact in the yeast two-hybrid system. In vivo labelling studies using a strain with a mutant allele of the prp4 gene in the genetic background indicate a change in phosphorylation of the Prp1p protein. These results are consistent with the notion that Prp4p kinase is involved in the control of the formation of active spliceosomes, targeting non-SR splicing factors

Overexpression of the <i>cwf16</i>^<i>+</i>, <i>srp2</i>^<i>+</i>, or <i>tif213</i>^<i>+</i> gene suppressed exon skipping in <i>ods4</i>-<i>1</i>.

<p>(A) The overexpression of the <i>cwf16</i>⁺ or <i>tif213</i>⁺ gene rescued the <i>cs</i> phenotype of <i>ods4-1</i>. Transformants with pBG1-URA4β and the <i>cwf16</i>^<i>+</i>, <i>srp2</i>^<i>+</i>, or <i>tif213</i>^<i>+</i> plasmid were streaked on MMU plates and incubated at 22, 26, or 30°C to test their complementarity for the <i>cs</i> phenotype of <i>ods4-1</i>. Transformants with the <i>cwf16</i>^<i>+</i> or <i>tif213</i>^<i>+</i> plasmid grew well at 22°C, whereas <i>ods4-1</i> itself showed slow growth at the same temperature (<i>ods4</i>+URA4β+pSP1). The overexpression of Srp2p resulted in slow growth at all temperatures. (B) Total RNAs were isolated from wild type (WT), <i>ods4-1</i>, and <i>ods4-1</i> with the <i>cwf16</i>^<i>+</i>, <i>srp2</i>^<i>+</i>, or <i>tif213</i>⁺ plasmid, and subjected to RT-PCR analyses. All transformants contained pBG1-URA4β in addition to the rescued genes or pSP1 vector as indicated. Amplified cDNA products were electrophoresed on a 5% acrylamide gel, stained with ethidium bromide (upper panel), and then subjected to a Southern blot analysis using an oligonucleotide probe that specifically hybridizes to the exon-skipped product (middle panel). RT-PCR of <i>act1</i>^<i>+</i> mRNA was performed as a control (lower panel). The structures of the RT-PCR products confirmed by the sequence analysis are shown on the left. Arrows indicate the positions of the tub-3 and tub-4 primers used for RT-PCR analyses.</p

Structure of reporter plasmids for <i>ods</i> screening.

<p>(A) Structures of pURA4β (pSP1-URA4β) and pBG1-URA4β reporter plasmids. The pSP1 and pBG1 vectors have the <i>LEU2</i> and <i>his3</i> markers, respectively. The intron 1-exon 2-intron 2 region of the <i>S</i>. <i>pombe</i> β-tubulin gene (<i>nda3</i>^<i>+</i>) was amplified by PCR and inserted into the <i>Stu</i> I site in the <i>ura4</i>^<i>+</i> gene. (B) Splicing patterns of the transcripts from the chimeric <i>ura4</i> gene in the reporter plasmids. Transcripts in which the internal exon is included produce non-functional Ura4p, whereas exon-skipped transcripts produce functional Ura4p.</p