Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein

In Saccharomyces cerevisiae, temperature-sensitive mutations in the genes RNA14 and RNA15 correlate with a reduction of mRNA stability and poly(A) tail length. Although mRNA transcription is not abolished in these mutants, the transcripts are rapidly deadenylated as in a strain carrying an RNA polymerase B(II) temperature-sensitive mutation. This suggests that the primary defect could be in the control of the poly(A) status of the mRNAs and that the fast decay rate may be due to the loss of this control. By complementation of their temperature-sensitive phenotype, we have cloned the wild-type genes. They are essential for cell viability and are unique in the haploid genome. The RNA14 gene, located on chromosome H, is transcribed as three mRNAs, one major and two minor, which are 2.2, 1.5, and 1.1 kb in length. The RNA15 gene gives rise to a single 1.2-kb transcript and maps to chromosome XVI. Sequence analysis indicates that RNA14 encodes a 636-amino-acid protein with a calculated molecular weight of 75,295. No homology was found between RNA14 and RNA15 or between RNA14 and other proteins contained in data banks. The RNA15 DNA sequence predicts a protein of 296 amino acids with a molecular weight of 32,770. Sequence comparison reveals an N-terminal putative RNA-binding domain in the RNA15-encoded protein, followed by a glutamine and asparagine stretch similar to the opa sequences. Both RNA14 and RNA15 wild-type genes, when cloned on a multicopy plasmid, are able to suppress the temperature-sensitive phenotype of strains bearing either the rna14 or the rna15 mutation, suggesting that the encoded proteins could interact with each other.

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The Last RNA-Binding Repeat of the Escherichia coliRibosomal Protein S1 Is Specifically Involved in Autogenous Control

Journal of Bacteriology ◽

10.1128/jb.182.20.5872-5879.2000 ◽

2000 ◽

Vol 182 (20) ◽

pp. 5872-5879 ◽

Cited By ~ 40

Author(s):

Irina V. Boni ◽

Valentina S. Artamonova ◽

Marc Dreyfus

Keyword(s):

Translation Initiation ◽

Rna Binding ◽

Binding Domain ◽

Start Codon ◽

Upstream Sequence ◽

Wild Type ◽

Gene Encoding ◽

Autogenous Control ◽

Extragenic Suppressor ◽

Rna Binding Domain

ABSTRACT The ssyF29 mutation, originally selected as an extragenic suppressor of a protein export defect, has been mapped within the rpsA gene encoding ribosomal protein S1. Here, we examine the nature of this mutation and its effect on translation. Sequencing of the rpsA gene from the ssyFmutant has revealed that, due to an IS10R insertion, its product lacks the last 92 residues of the wild-type S1 protein corresponding to one of the four homologous repeats of the RNA-binding domain. To investigate how this truncation affects translation, we have created two series of Escherichia coli strains (rpsA + and ssyF) bearing various translation initiation regions (TIRs) fused to the chromosomallacZ gene. Using a β-galactosidase assay, we show that none of these TIRs differ in activity between ssyF andrpsA + cells, except for the rpsATIR: the latter is stimulated threefold in ssyF cells, provided it retains at least ca. 90 nucleotides upstream of the start codon. Similarly, the activity of this TIR can be severely repressed in trans by excess S1, again provided it retains the same minimal upstream sequence. Thus, the ssyFstimulation requires the presence of the rpsA translational autogenous operator. As an interpretation, we propose that thessyF mutation relieves the residual repression caused by normal supply of S1 (i.e., that it impairs autogenous control). Thus, the C-terminal repeat of the S1 RNA-binding domain appears to be required for autoregulation, but not for overall mRNA recognition.

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Immunogenicity and Protection Efficacy of Replication-Deficient Influenza A Viruses with Altered NS1 Genes

Journal of Virology ◽

10.1128/jvi.78.23.13037-13045.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13037-13045 ◽

Cited By ~ 85

Author(s):

Boris Ferko ◽

Jana Stasakova ◽

Julia Romanova ◽

Christian Kittel ◽

Sabine Sereinig ◽

...

Keyword(s):

Influenza A ◽

Rna Binding ◽

T Cell Response ◽

Binding Domain ◽

Wild Type Virus ◽

Type Virus ◽

Ns1 Protein ◽

Wild Type ◽

Influenza A Viruses ◽

Rna Binding Domain

ABSTRACT We explored the immunogenic properties of influenza A viruses with altered NS1 genes (NS1 mutant viruses). NS1 mutant viruses expressing NS1 proteins with an impaired RNA-binding function or insertion of a longer foreign sequence did not replicate in murine lungs but still were capable of inducing a Th1-type immune response resulting in significant titers of virus-specific serum and mucosal immunoglobulin G2 (IgG2) and IgA, but with lower titers of IgG1. In contrast, replicating viruses elicited high titers of serum and mucosal IgG1 but less serum IgA. Replication-deficient NS1 mutant viruses induced a rapid local release of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-6. Moreover, these viruses also elicited markedly higher levels of IFN-α/β in serum than the wild-type virus. Comparable numbers of virus-specific primary CD8+ T cells were determined in all of the groups of immunized mice. The most rapid onset of the recall CD8+-T-cell response upon the wild-type virus challenge was detected in mice primed with NS1 mutant viruses eliciting high levels of cytokines. It is noteworthy that there was one NS1 mutant virus encoding NS1 protein with a deletion of 40 amino acids predominantly in the RNA-binding domain that induced the highest levels of IFN-α/β, IL-6 and IL-1β after infection. Mice that were immunized with this virus were completely protected from the challenge infection. These findings indicate that a targeted modification of the RNA-binding domain of the NS1 protein is a valuable technique to generate replication-deficient, but immunogenic influenza virus vaccines.

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Sequence analysis of Indian SARS-CoV-2 isolates shows a stronger interaction of mutated receptor binding domain with ACE2 receptor

10.1101/2020.08.28.271601 ◽

2020 ◽

Author(s):

Pujarini Dash ◽

Jyotirmayee Turuk ◽

Santosh Ku. Behera ◽

Subrata Ku. Palo ◽

Sunil K. Raghav ◽

...

Keyword(s):

Sequence Analysis ◽

Binding Affinity ◽

Reference Strain ◽

Rna Binding ◽

Throat Swab ◽

Binding Domain ◽

Spike Protein ◽

Family Welfare ◽

Eastern India ◽

Wild Type

AbstractSARS-CoV-2 is a RNA Coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). It has affected the whole world including Odisha, a state in eastern India. Many people migrated in the state from different countries as well as states during this SARS-CoV-2 pandemic. As per the protocol laid by ICMR and Health & Family welfare of India, all the suspected cases were tested for SARS-CoV-2 infection. The aim of this study was to analyze the RNA binding domain (RBD) sequence of spike protein from the isolates collected from the throat swab samples of COVID-19 positive cases and further to assess the RBD affinity with ACE2 of different species including human.Whole genome sequencing for 35 clinical SARS-CoV-2 isolates from COVID-19 positive patients was performed using ARTIC amplicon based sequencing. Sequence analysis and phylogenetic analysis was carried out for the Spike and RBD region of all isolates. The interaction between the RBD and ACE2 receptor of five different species was also analysed.Except three isolates, spike region of 32 isolates showed one/multiple alterations in nucleotide bases in comparison to the Wuhan reference strain. One of the identified mutation at 1204 (Ref A, RMRC 22 C) in the RBD of spike protein was identified which depicted a stronger binding affinity with human ACE2 receptor compared to the wild type RBD. Furthermore, RBDs of all the Indian isolates are capable of binding to ACE2 of human, bat, hamster and pangolin.As mutated RBD showed stronger interaction with human ACE2, it could potentially result in higher infectivity. The study shows that RBDs of all the studied isolates have binding affinity for all the five species, which suggests that the virus can infect a wide variety of animals which could also act as natural reservoir for SARS-CoV-2.

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