Reading m6A in the Transcriptome: m6A-Binding Proteins.

N6-Methyladenosine (m6A) is the most prevalent post-transcriptional modification of eukaryotic mRNA and long noncoding RNA. m6A mediates its effects primarily by recruiting proteins, including the multiprotein eukaryotic initiation factor 3 complex and a set of proteins that contain the YTH domain. Here we describe the mechanisms by which YTH domain-containing proteins bind m6A and influence the fate of m6A-containing RNA in mammalian cells. We discuss the diverse, and occasionally contradictory, functions ascribed to these proteins and the emerging concepts that are influencing our understanding of these proteins and their effects on the epitranscriptome.

Potential Role of Hepatitis C Virus Alternate Reading Frame Protein in Negative Regulation of T-Bet Gene Expression.

Hepatitis C virus (HCV) is a major cause of chronic liver disease and has led to cirrhosis or hepatocellular carcinoma in a majority of infected individuals. We have previously demonstrated that the HCV alternate reading frame protein (F protein) is related to Th1/Th2 bias in chronic hepatitis C (CHC) patients, and we aimed to explore the relative molecular mechanisms here. A total of 104 cases including CHC patients and healthy donors were enrolled. T-bet and GATA-3 expression levels were analyzed in peripheral blood mononuclear cells (PBMCs). The levels of signal transducer and activator of transcription-1/-6(STAT1/6) and phosphorylated STAT1/6(pSTAT1/6) in PBMCs were measured by Western blotting. Our results showed that the levels of T-bet in PBMCs, as well as the levels of gamma interferon (IFN-γ) in sera, were decreased in anti-F protein antibody seropositive patients compared with anti-F protein antibody seronegative patients, whereas the levels of GATA-3 did not show difference between the two groups. Moreover, the decreased pSTAT1 and increased pSTAT6 were observed in PBMCs by HCV core/F protein stimulation with constant STAT1/6 expression. Taken together, it suggested that T-bet may be involved in Th1/Th2 bias induced by HCV F protein, and the disruption of STAT phosphorylation may participate in this mediation.

An extended genetic scale of reading frame coding.

The reading frame coding (RFC) of codes (sets) of trinucleotides is a genetic concept which has been largely ignored during the last 50 years. An extended definition of the statistical parameter PrRFC (Michel, 2014) is proposed here for analysing the probability (efficiency) of reading frame coding of usage of any trinucleotide code. It is applied to the analysis of the RFC efficiency of usage of the C(3) self-complementary trinucleotide circular code X identified in prokaryotic and eukaryotic genes (Arquès and Michel, 1996). The usage of X is called usage XU. The highest RFC probabilities of usage XU are identified in bacterial plasmids and bacteria (about 49.0%). Then, by decreasing values, the RFC probabilities of usage XU are observed in archaea (47.5%), viruses (45.4%) and nuclear eukaryotes (42.8%). The lowest RFC probabilities of usage XU are found in mitochondria and chloroplasts (about 36.5%). Thus, genes contain information for reading frame coding. Such a genetic property which to our knowledge has never been identified, may bring new insights in the origin and evolution of the genetic code.

Inter-dinucleotide distances in the human genome: an analysis of the whole-genome and protein-coding distributions.

We study the inter-dinucleotide distance distributions in the human genome, both in the whole-genome and protein-coding regions. The inter-dinucleotide distance is defined as the distance to the next occurrence of the same dinucleotide. We consider the 16 sequences of inter-dinucleotide distances and two reading frames. Our results show a period-3 oscillation in the protein-coding inter-dinucleotide distance distributions that is absent from the whole-genome distributions. We also compare the distance distribution of each dinucleotide to a reference distribution, that of a random sequence generated with the same dinucleotide abundances, revealing the CG dinucleotide as the one with the highest cumulative relative error for the first 60 distances. Moreover, the distance distribution of each dinucleotide is compared to the distance distribution of all other dinucleotides using the Kullback-Leibler divergence. We find that the distance distribution of a dinucleotide and that of its reversed complement are very similar, hence, the divergence between them is very small. This is an interesting finding that may give evidence of a stronger parity rule than Chargaff's second parity rule.

Regulated readthrough: a new method for the alternative tagging and targeting of recombinant proteins.

We report here a new method for the alternative peptide tagging of recombinant proteins from mammalian cell lines. This method, which we called regulated readthrough, exploits the property of aminoglycoside antibiotics to promote translational readthrough of nonsense codons. The basic expression cassette includes a translational fusion between a gene of interest and a membrane targeting peptide, which are separated by a nonsense codon. In the presence of an aminoglycoside antibiotic, translational readthrough is promoted and results in the targeting of the fusion protein to the cell membrane, thus allowing the efficient flow cytometry-based isolation of cells expressing very high levels of recombinant protein. For downstream applications requiring the production of soluble recombinant protein, the cells are cultured in the absence of aminoglycoside, leading to an efficient translational termination. By combining different translation termination signals that exhibit various susceptibilities to aminoglycoside-mediated translational readthrough with flow cytometry capabilities, it is possible to use this technology for other applications such as functional library screening or monitoring the stability of recombinant protein production.

Cuando la traducción produce diferencias: procesamiento de frases en lectura y traducción / When translation makes the difference: sentence processing in reading and translation

El artículo presenta datos de dos experimentos en que se compara la lectura normal y la lectura para traducir. En ambos Experimentos las frases se presentaban palabra a palabra. En el Experimento 1, un grupo de traductores profesionales leían frases con instrucciones de repetirlas en español o traducirlas al inglés. Además manipulamos la disponibilidad de información pragmática en las oraciones. En el Experimento 2 se invirtió la lengua de origen y se pedía a los traductores que repitiesen las frases en inglés después de su lectura o que las tradujesen al castellano. En los dos experimentos los tiempos de lectura fueron más lentos en la lectura para traducir que en la lectura para repetir, lo que indica que la comprensión de frases es dependiente del objetivo de la lectura. La presencia de información pragmática facilitaba la comprensión, pero sólo cuando el idioma origen era el castellano. Esta asimetría entre idiomas indica que la utilización de pistas pragmáticas para facilitar la lectura puede depender del idioma en el que se lee. Los resultados en su conjunto proporcionan apoyo a las teorías horizontales de traducción (AU)

P-site tRNA is a crucial initiator of ribosomal frameshifting.

The expression of some genes requires a high proportion of ribosomes to shift at a specific site into one of the two alternative frames. This utilized frameshifting provides a unique tool for studying reading frame control. Peptidyl-tRNA slippage has been invoked to explain many cases of programmed frameshifting. The present work extends this to other cases. When the A-site is unoccupied, the P-site tRNA can be repositioned forward with respect to mRNA (although repositioning in the minus direction is also possible). A kinetic model is presented for the influence of both, the cognate tRNAs competing for overlapping codons in A-site, and the stabilities of P-site tRNA:mRNA complexes in the initial and new frames. When the A-site is occupied, the P-site tRNA can be repositioned backward. Whether frameshifting will happen depends on the ability of the A-site tRNA to subsequently be repositioned to maintain physical proximity of the tRNAs. This model offers an alternative explanation to previously published mechanisms of programmed frameshifting, such as out-of-frame tRNA binding, and a different perspective on simultaneous tandem tRNA slippage.

Frame switch splicing and regulated intramembrane proteolysis: key words to understand the unfolded protein response.

Proteins must be correctly folded and assembled to fulfill their functions as assigned by genetic code. All living cells have developed systems to counteract protein unfolding or misfolding. A typical example of such a homeostatic response is triggered when unfolded proteins are accumulated in the endoplasmic reticulum. Eukaryotic cells cope with endoplasmic reticulum stress by attenuating translation, generally to decrease the burden on the folding machinery, as well as by inducing transcription of endoplasmic reticulum-localized molecular chaperones and folding enzymes to augment folding capacity. These translational and transcriptional controls are collectively termed the unfolded protein response. The unfolded protein response is unique in that the molecular mechanisms it uses to transmit signals from the endoplasmic reticulum lumen to the nucleus are completely different from those used for signaling from the plasma membrane. Frame switch splicing (a term newly proposed here) and regulated intramembrane proteolysis (proposed by Brown et al., Cell 2000; 100: 391-398) employed by the unfolded protein response represent novel ways to activate a signaling molecule post-transcriptionally and post-translationally, respectively. They are critically involved in various cellular regulation pathways ranging from bacterial extracytoplasmic stress response to differentiation of mature B cells into antibody-secreting plasma cells. Further, mammalian cells take advantage of differential properties between the two mechanisms to determine the fate of proteins unfolded or misfolded in the endoplasmic reticulum. This review focuses on the transcriptional control that occurs during the unfolded protein response in various species.

Role of the alternating reading frame (P19)-p53 pathway in an in vivo murine colon tumor model.

Considering the importance of the oncogene checkpoint function of the alternating reading frame(ARF)-p53 pathway, studies were undertaken to evaluate the status of this pathway in azoxymethane (AOM)-induced mouse colon tumors. A PCR-based analysis of ARF and p53 cDNAs in normal colon tissues and AOM-induced colon tumors failed to detect mutations in either of these two critical tumor suppressor genes. In addition, laser capture microdissection of tumors followed by PCR-based sequencing of exons 5-7 of genomic p53 showed that even the most pleomorphic cancer cells were p53 normal. A marked increase in ARF mRNA and protein levels was observed in colon tumors, indicating activation of the ARF-p53 pathway in these tumors. High levels of ARF protein stabilized p53 protein in the tumors, but the p53 protein showed little biochemical activity. Compared with a mouse colonocyte cell line that expresses high levels of wild-type p53 (YAMC), the p53 protein in tumors had no detectable DNA binding activity nor did it activate p21 expression. In fact, p21 levels were lower in tumor tissue relative to normal mucosa, even though p53 levels were approximately 30-fold higher in tumors relative to control. Within the A/J tumors, we also used a cDNA microarray approach to screen a panel of genes that are transcriptionally up- or down-regulated by functional p53. The expression patterns of these p53-regulated genes were consistent with a lack of functional p53. This work demonstrates that the ARF-p53 oncogene checkpoint can be overcome without p53 mutations and that the mechanism used to overcome this checkpoint involves the suppression of p53 transcriptional activating activity. The AOM colon cancer model may be well suited for studying tumor promotion events that precede p53 disruption.

Procesamiento morfológico en el reconocimiento de palabras: una revisión con especial referencia a los datos en español / Morphological processing in word recognition: a review with particular reference to Spanish data

El objetivo de este artículo es presentar de forma organizada los resultados que apoyan el procesamiento de las unidades morfológicas de palabras aisladas desde distintos paradigmas experimentales. Para ello se han revisado tres hipótesis que proponen distintas soluciones al problema del papel de la morfología en el acceso al léxico: a) segmentación obligatoria, b) listado exhaustivo y c) hipótesis mixta. Se barajan los problemas y ventajas de cada una de ellas, y de los modelos que representan, a la luz de los datos que provienen de las siguientes manipulaciones experimentales: pseudopalabras estructuradas morfológicamente, palabras monomorfémicas versus palabras polimorfémicas, estudios de priming morfológico y comparación entre frecuencia acumulada y frecuencia superficial (AU)

Influencia de variables léxicas y subléxicas en adultos neolectores / Influence of lexical and sublexical in adults illiterates

El primer objetivo de nuestro estudio era encontrar el efecto de variables léxicas y subléxicas (categoría léxica, frecuencia léxica, categoría gramatical y longitud de la palabra) en la adquisición de la lectura en adultos neolectores en la adquisición de una lengua transparente como el castellano. El segundo objetivo era comparar el efecto de esas variables en adultos neolectores que se inician en el aprendizaje de la lectura y aquéllos que llevan más de tres años aprendiendo. Usamos cuarenta y cuatro adultos (con edades comprendidas entre los 42 y 72 años), divididos en dos grupos de veintidós personas cada uno según los años que llevaban en el centro aprendiendo a leer. Todas los sujetos realizaron una prueba de lectura con 306 ítems en la cual estaban contrabalanceadas todas las variables anteriormente mencionadas. La variable dependiente era el porcentaje de respuestas correctas en la prueba de lectura. Los resultados mostraron que había diferencias significativas en el número de errores cometidos entre los dos grupos de adultos. Las cuatro variables estudiadas mostraron el mismo tipo de efecto sobre la ejecución lectora, tanto para los sujetos que acaban de comenzar a leer, como y aquéllos que llevaban más tiempo, indicando que los primeros utilizan la ruta léxica y la fonológica. Los datos sugieren la universalidad del modelo de la doble ruta. (AU)

An rRNA fragment and its antisense can alter decoding of genetic information.

rRNA plays a central role in protein synthesis and is intimately involved in the initiation, elongation, and termination stages of translation. However, the mode of its participation in these reactions, particularly as to the decoding of genetic information, remains elusive. In this paper, we describe a new approach that allowed us to identify an rRNA segment whose function is likely to be related to translation termination. By screening an expression library of random rRNA fragments, we identified a fragment of the Escherichia coli 23S rRNA (nucleotides 74 to 136) whose expression caused readthrough of UGA nonsense mutations in certain codon contexts in vivo. The antisense RNA fragment produced a similar effect, but in neither case was readthrough of UAA or UAG observed. Since termination at UGA in E. coli specifically requires release factor 2 (RF2), our data suggest that the fragments interfere with RF2-dependent termination.