Leader Proteinase of the Beet Yellows Closterovirus: Mutation Analysis of the Function in Genome Amplification

ABSTRACT The beet yellows closterovirus leader proteinase (L-Pro) possesses a C-terminal proteinase domain and a nonproteolytic N-terminal domain. It was found that although L-Pro is not essential for basal-level replication, deletion of its N-terminal domain resulted in a 1,000-fold reduction in RNA accumulation. Mutagenic analysis of the N-terminal domain revealed its structural flexibility except for the 54-codon-long, 5′-terminal element in the corresponding open reading frame that is critical for efficient RNA amplification at both RNA and protein levels.

Download Full-text

att, a target for regulation by tra2 in the testes of Drosophila melanogaster, encodes alternative RNAs and alternative proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.4222 ◽

1996 ◽

Vol 16 (8) ◽

pp. 4222-4230 ◽

Cited By ~ 16

Author(s):

S J Madigan ◽

P Edeen ◽

J Esnayra ◽

M McKeown

Keyword(s):

Rna Processing ◽

Transmembrane Domain ◽

Somatic Tissue ◽

Open Reading Frame ◽

Graves Disease ◽

Carrier Protein ◽

Reading Frame ◽

Protein Levels ◽

Somatic Tissues ◽

Promoter Usage

We have identified a gene, alternative testis transcripts (att), which is alternatively expressed, at both the RNA and protein levels, in testes and somatic tissues. The testis-specific RNA differs from somatic RNAs in both promoter usage and RNA processing and is dependent on the function of the transformer 2 gene. The differences between the somatic and testis RNAs have substantial consequences at the protein level. The somatic RNAs encode a protein with homology to the mammalian Graves' disease carrier proteins. The testis RNA lacks the initiation codons used in somatic tissue and encodes two different proteins. One of these begins in a testis-specific exon, uses a reading frame different from that for the somatic protein, and is completely novel. The other protein initiates translation in the frame of the somatic RNA at a Len CUG codon which is within the open reading frame for the somatic protein. This produces a novel truncated version of the Graves' disease carrier protein-like protein that lacks all sequences N terminal to the first transmembrane domain.

Download Full-text

Open reading frame correction using antisense oligonucleotides for the treatment of cystic fibrosis caused by CFTR-W1282X

10.1101/2021.08.11.455834 ◽

2021 ◽

Author(s):

Wren E. Michaels ◽

Cecilia Pena-Rasgado ◽

Rusudan Kotaria ◽

Robert J. Bridges ◽

Michelle L. Hastings

Keyword(s):

Cystic Fibrosis ◽

Protein Function ◽

Antisense Oligonucleotides ◽

Gene Mutations ◽

Severe Form ◽

Open Reading Frame ◽

Common Mutation ◽

Reading Frame ◽

Protein Levels ◽

Cftr Protein

CFTR gene mutations that result in the introduction of premature termination codons (PTCs) are common in cystic fibrosis (CF). This mutation type causes a severe form of the disease, likely because of low CFTR mRNA expression as a result of nonsense mediated mRNA decay (NMD), as well as production of a non-functional, truncated CFTR protein. Current therapeutics for CF, which target residual protein function, are less effective in patients with these types of mutations, due in part to low CFTR protein levels. Splice-switching antisense oligonucleotides (ASOs) designed to induce skipping of exons in order to restore the mRNA open reading frame have shown therapeutic promise pre-clinically and clinically for a number of diseases. We hypothesized that ASO-mediated skipping of CFTR exon 23 would recover CFTR activity associated with terminating mutations in the exon, including CFTR p.W1282X, the 5th most common mutation in CF. Here, we show that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use. ASO-induced exon 23 skipping rescued CFTR expression and chloride current in primary human bronchial epithelial cells isolated from homozygote CFTR-W1282X patients. These results support the use of ASOs in treating CF patients with CFTR class I mutations in exon 23 that result in unstable CFTR mRNA and truncations of the CFTR protein.

Download Full-text

Quantitating Translational Control: mRNA Abundance-Dependent and Independent Contributions and the mRNA Sequences That Specify Them

10.1101/116913 ◽

2017 ◽

Cited By ~ 1

Author(s):

Jingyi Jessica Li ◽

Guo-Liang Chew ◽

Mark D. Biggin

Keyword(s):

Translational Control ◽

Mrna Level ◽

Open Reading Frame ◽

Mrna Abundance ◽

Mrna Levels ◽

Mrna Sequence ◽

Translation Rate ◽

Reading Frame ◽

Protein Levels ◽

Selective Pressures

AbstractTranslation rate per mRNA molecule correlates positively with mRNA abundance. As a result, protein levels do not scale linearly with mRNA levels, but instead scale with the abundance of mRNA raised to the power of an “amplification exponent”. Here we show that to quantitate translational control, the translation rate must be decomposed into two components. One, TRmD, depends on the mRNA level and defines the amplification exponent. The other, TRmIND, is independent of mRNA amount and impacts the correlation coefficient between protein and mRNA levels. We show that in S. cerevisiae TRmD represents ∼20% of the variance in translation and directs an amplification exponent of 1.20 with a 95% confidence interval [1.14, 1.26]. TRmIND constitutes the remaining ∼80% of the variance in translation and explains ∼5% of the variance in protein expression. We also find that TRmD and TRmIND are preferentially determined by different mRNA sequence features: TRmIND by the length of the open reading frame and TRmD both by a ∼60 nucleotide element that spans the initiating AUG and by codon and amino acid frequency. Our work provides more appropriate estimates of translational control and implies that TRmIND is under different evolutionary selective pressures than TRmD.

Download Full-text

The 2b Protein of Cucumoviruses Has a Role in Promoting the Cell-to-cell Movement of Pseudorecombinant Viruses

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2003.16.3.261 ◽

2003 ◽

Vol 16 (3) ◽

pp. 261-267 ◽

Cited By ~ 38

Author(s):

Bu-Jun Shi ◽

Jane Miller ◽

Robert H. Symons ◽

Peter Palukaitis

Keyword(s):

Amino Acids ◽

Specific Interaction ◽

Cell Movement ◽

Open Reading Frame ◽

In Planta ◽

Reading Frame ◽

Tomato Aspermy Virus ◽

2B Protein ◽

Rna Accumulation

Pseudorecombinant viruses (i.e., those containing a reas-sorted genome of closely related multipartite viruses) are often not as competitive as the parental viruses. The role of the 2b gene in hypervirulence and maintenance of a progressive infection was assessed in a pseudorecombinant virus formed between RNAs 1 plus 2 of Cucumber mosaic virus (CMV) and RNA 3 of Tomato aspermy virus (TAV). The presence of RNA 3 of TAV was found to affect the level of RNA accumulation but not the level of virulence. By contrast, the 2b genes of both TAV and a hypervirulent strain of CMV (WAII-CMV) were found to affect the virulence of the pseudorecombinant viruses but not the levels of viral RNA accumulation. The 2b gene rather than the overlapping open reading frame encoding the C-terminal 41 amino acids of 2a protein of the corresponding virus was found to be essential for promoting infection of the pseudorecombinant viruses in planta. However, the 2b gene was not essential for replication of pseudorecombinant viruses containing CMV RNAs 1 plus 2 and TAV RNA 3. These results indicate that the 2b protein is involved in promoting the cell-to-cell movement of the pseudorecombinant viruses. These data also suggest the existence of specific interaction between the TAV 2b protein and either RNA 3 or its encoded proteins, which may be critical for promoting or maintaining infection or both.

Download Full-text

Interaction between the Open Reading Frame III Product and the Coat Protein Is Required for Transmission of Cauliflower Mosaic Virus by Aphids

Journal of Virology ◽

10.1128/jvi.75.1.100-106.2001 ◽

2001 ◽

Vol 75 (1) ◽

pp. 100-106 ◽

Cited By ~ 32

Author(s):

Véronique Leh ◽

Emmanuel Jacquot ◽

Angèle Geldreich ◽

Muriel Haas ◽

Stéphane Blanc ◽

...

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Direct Interaction ◽

Cauliflower Mosaic Virus ◽

Open Reading Frame ◽

Nonstructural Proteins ◽

Interaction Domain ◽

Reading Frame ◽

Virus Particles ◽

Terminal Domain

ABSTRACT Transmission of cauliflower mosaic virus (CaMV) by aphids requires two viral nonstructural proteins, the open reading frame (ORF) II and ORF III products (P2 and P3). An interaction between a C-terminal domain of P2 and an N-terminal domain of P3 is essential for transmission. Purified particles of CaMV are efficiently transmitted only if aphids, previously fed a P2-containing solution, are allowed to acquire a preincubated mixture of P3 and virions in a second feed, thus suggesting a direct interaction between P3 and coat protein. Herein we demonstrate that P3 directly interacts with purified viral particles and unassembled coat protein without the need for any other factor and that P3 mediates the association of P2 with purified virus particles. The interaction domain of P3 is located in its C-terminal half, downstream of the P3-P2 interaction domain but overlapping a region which binds nucleic acids. Mutagenesis of P3 which interferes with the interaction between P3 and virions is correlated with the loss of transmission by aphids. Taken together, our results demonstrate that P3 plays a crucial role in the formation of the CaMV transmissible complex by serving as a bridge between P2 and virus particles.

Download Full-text