A plant virus replication system to assay the formation of RNA pseudotriloop motifs in RNA–protein interactions

Abstract

A pseudotriloop is formed by transloop base pairing between the first (5 ) and the fifth nucleotide in a hexanucleotide RNA loop (''hexaloop'') to subtend a triloop of nucleotides 2-4. This structure has been found in hairpins involved in the regulation of iron metabolism in mammalian cells and in transcription of plant virus subgenomic RNA. Several hexaloop hairpins, including HIV-transactivation-responsive element and hepatitis B virus , potentially adopt a pseudotriloop conformation. Here we show that an RNA plant virus whose replication depends on a conventional triloop hairpin can be used to verify the existence of pseudotriloop structures in vivo. Our data suggest that the pseudotriloop may represent a common motif in RNA-protein recognition.R NA-protein interactions play a fundamental role in various cellular processes and viral infections. Unlike DNA-binding proteins, which recognize sequence-specific motifs, proteins that interact with RNA often recognize structural features such as hairpin loops, bulges, and internal loops. The number of solved RNA-protein complexes has grown dramatically in recent years and has provided insight into the mechanisms of RNA recognition (1, 2). However, RNA motifs inferred from these studies are usually restricted to a specific class of related proteins and are not widely applicable.The family Bromoviridae comprises plant viruses with a tripartite RNA genome of messenger polarity (Fig. 1A). RNAs 1 and 2 encode the viral subunits of the RNA-dependent RNA polymerase (RdRp). The dicistronic RNA 3 encodes the movement protein (P3) and coat protein (CP). The latter is translated from a subgenomic (sg) mRNA 4. The best-studied members of this family are alfalfa mosaic virus (AMV) and brome mosaic virus (BMV) (3, 4).For BMV we found that in vitro sgRNA synthesis relies on the presence of a small hairpin with a CAUAGA loop, but that a conventional trinucleotide ''triloop''-terminated hairpin could functionally replace this hexaloop-terminated hairpin structure (5). Using biochemical and biophysical techniques, we demonstrated that the CAUAGA loop adopts a triloop conformation by transloop base pairing of C1 and G5, creating a AUA triloop and bulging out A6. This conformation, which we have termed ''pseudotriloop'' (PTL), resembles the structure of iron-responsive elements (IREs) that regulate translation of mRNAs encoding proteins involved in iron uptake, storage, and utilization in mammalian cells. In the conserved loop motif CAGUGH (H ϭ A, C, or U) of IREs, a similar transloop base pair between C1 and G5 is essential for recognition by iron-regulatory proteins (6 -9). In contrast to the IRE loop, deletion of the bulge (A6) in the BMV loop was not detrimental for subgenomic promoter (sgp) activity. This finding suggested that the BMV RdRp is able to use both triloop and PTL hairpins for sg transcription (5).We demonstrated (10) that AMV RdRp also recognizes a triloop hairpin to direct sgRNA synthesis in vitro (Fig. 1B). Given its relatedness to BMV, we anticipated that AMV RdRp ...

Structural and thermodynamic signatures that define pseudotriloop RNA hairpins

“…The HIV-1 TAR hairpin has been studied using NMR by others before, but in these cases the CG cross-loop resonance was also not observed (Jaeger and Tinoco 1993;Aboul-Ela et al 1996;Dethoff et al 2008Dethoff et al , 2012 although there is functional data to support its presence (Haasnoot et al 2003;Kulinski et al 2003). Our NMR data also showed no indication of a cross-loop base pair in tHIV and we ascribe this to the presence of an A-bulge and loop-closing CG base pair.…”

“…We investigated the existence and thermodynamic stability of several PTL structures derived from various viral sources, including the HBV ɛ and HIV-1 TAR loops, and two postulated candidate PTLs: a stem-loop from a spumaretrovirus (HFV) and a bacterial hairpin involved in regulation of pyrimidine synthesis, pyrR (Haasnoot et al 2003).…”

“…To support this view, it was recently demonstrated that the PTL and triloop promoter hairpins of brome mosaic virus (BMV) adopt an almost identical structure, although the latter one has higher thermodynamic stability (Skov et al 2012). It has been shown that PTLs are recognition sites for proteins (Haasnoot et al 2000(Haasnoot et al , 2002(Haasnoot et al , 2003: Disruption of the cross-loop base pair diminishes or sometimes even abolishes protein binding, and thus affects functioning of the RNA.…”

The subgenomic promoter of brome mosaic virus folds into a stem–loop structure capped by a pseudo-triloop that is structurally similar to the triloop of the genomic promoter

“…In this respect, BMV does not differ from the related alfalfa mosaic virus (AMV), for which it has been shown that the genomic promoter could substitute the subgenomic promoter in vitro and in vivo (Olsthoorn et al 2004). Interestingly, the AMV promoters for sgRNA and gRNA synthesis form a hairpin with a regular triloop but can be functionally replaced by hairpins with a pseudo-triloop, like HBV e (Flodell et al 2002(Flodell et al , 2006Haasnoot et al 2003), HIV-1 TAR (Haasnoot et al 2003) and the BMV subgenomic promoter (RCL Olsthoorn, unpubl. ).…”

A comparative analysis of the triloops in all high-resolution RNA structures reveals sequence–structure relationships

“…The triloop motif is particularly interesting due to its major role in a variety of organisms and pathways, for instance, in the promotion of some virus replication (Huang et al 2001;Olsthoorn and Bol 2002), viral synthesis (Haasnoot et al 2003), and iron response (McCallum and Pardi 2003). Furthermore, a previous triloop study revealed an important structural diversity (Lee et al 2003), making this motif an ideal candidate for a base interaction characterization.…”