Testing of internal translation initiation via dicistronic constructs in yeast is complicated by production of extraneous transcripts

Mäkeläinen, Katri; Mäkinen, Kristiina

doi:10.1016/j.gene.2007.01.010

Cited by 11 publications

(8 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A transformation-based assay for IRES activities requires validation because IRES activities detected in such assays may represent those from spliced or degraded transcripts or transcripts from a cryptic promoter with the OFluc ORF situated at the most 5′-proximal end rather than from dicistronic dual-luciferase transcripts ( 6 , 34 ). To circumvent these problems ( 35 ), we took an RNA electroporation approach where C. parasitica spheroplasts were transfected with in vitro -synthesized RNA from representative constructs.…”

Section: Resultsmentioning

confidence: 99%

First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes

Chiba

Jamal

Suzuki

2018

mBio

View full text Add to dashboard Cite

In contrast to well-established internal ribosomal entry site (IRES)-mediated translational initiation in animals and plants, no IRESs were established in fungal viral or cellular RNAs. To identify IRES elements in mycoviruses, we developed a luciferase-based dual-reporter detection system in Cryphonectria parasitica, a model filamentous fungus for virus-host interactions. A bicistronic construct entails a codon-optimized Renilla and firefly luciferase (ORluc and OFluc, respectively) gene, between which potential IRES sequences can be inserted. In this system, ORluc serves as an internal control, while OFluc represents IRES activity. Virus sequences in the 5′ untranslated regions (UTRs) of the genomes of diverse positive-sense single-stranded RNA and double-stranded RNA (dsRNA) viruses were analyzed. The results show relatively high IRES activities for Cryphonectria hypovirus 1 (CHV1) and CHV2 and faint but measurable activity for CHV3. The weak IRES signal of CHV3 may be explained by its monocistronic nature, differing from the bicistronic nature of CHV1 and CHV2. This would allow these three hypoviruses to have similar rates of translation of replication-associated protein per viral mRNA molecule. The importance of 24 5′-proximal codons of CHV1 as well as the 5′ UTR for IRES function was confirmed. Furthermore, victoriviruses and chrysoviruses tested IRES positive, whereas mycoreoviruses, partitiviruses, and quadriviruses showed similar Fluc activities as the negative controls. Overall, this study represents the first development of an IRES identification system in filamentous fungi based on the codon-optimized dual-luciferase assay and provides evidence for IRESs in filamentous fungi.

show abstract

Section: Resultsmentioning

confidence: 99%

First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes

Chiba

Jamal

Suzuki

2018

mBio

View full text Add to dashboard Cite

show abstract

“…Generation of unwanted aberrant transcripts by cryptic splicing sites and/or cryptic promoters present in plasmid backbone and their ability to affect the particular assays also have been reported (Boshart et al 1992;Rosfjord et al 1994;Hennecke et al 2001;Hall et al 2002;Giannakis et al 2003;Van Eden et al 2004;Holcik et al 2005;Kozak, 2007;Makelainen and Makinen 2007). However, no significant attention has been paid to cryptic transcription from reporter genes until now.…”

Section: Introductionmentioning

confidence: 98%

Firefly luciferase gene contains a cryptic promoter

Vopálenský¹,

Mašek²,

Horváth³

et al. 2008

RNA

View full text Add to dashboard Cite

A firefly luciferase (FLuc) counts among the most popular reporters of present-day molecular and cellular biology. In this study, we report a cryptic promoter activity in the luc+ gene, which is the most frequently used version of the firefly luciferase. The FLuc coding region displays cryptic promoter activity both in mammalian and yeast cells. In human CCL13 and Huh7 cells, cryptic transcription from the luc+ gene is 10-16 times weaker in comparison to the strong immediate-early cytomegalovirus promoter. Additionally, we discuss a possible impact of the FLuc gene cryptic promoter on experimental results especially in some fields of the RNA-oriented research, for example, in analysis of translation initiation or analysis of miRNA/siRNA function. Specifically, we propose how this newly described cryptic promoter activity within the FLuc gene might contribute to the previous determination of the strength of the cryptic promoter found in the cDNA corresponding to the hepatitis C virus internal ribosome entry site. Our findings should appeal to the researchers to be more careful when designing firefly luciferasebased assays as well as open the possibility of performing some experiments with the hepatitis C virus internal ribosome entry site, which could not be considered until now.

show abstract

“…However, a positive result in a bicistronic test does not by itself demonstrate IRES activity. Over time, the reliability of this approach has been challenged by the detection of cryptic promoters or cryptic splicing sites in the genomic regions containing an IRES [5][6][7][8][9]. Aberrant transcripts can also stem from plasmid backbones by cryptic transcription [10][11][12][13] or by a unique combination of vector and IRES sequences that can induce cryptic splicing, producing monocistronic mRNAs that can pollute that particular assay [14][15][16][17].…”

Section: Introductionmentioning

confidence: 99%