Sequence requirements for localization and packaging of Ty3 retroelement RNA

Abstract

Retroviruses and retrotransposons package genomic RNA into virus-like particles (VLPs) in a poorly understood process. Expression of the budding yeast retrotransposon Ty3 results in the formation of cytoplasmic Ty3 VLP assembly foci comprised of Ty3 RNA and proteins, and cellular factors associated with RNA processing body (PB) components, which modulate translation and effect nonsense-mediated decay (NMD). A series of Ty3 RNA variants were tested to understand the effects of read-through translation via programmed frameshifting on RNA localization and packaging into VLPs, and to identify the roles of coding and non-coding sequences in those processes. These experiments showed that a low level of read-through translation of the downstream open reading frame (as opposed to no translation or translation without frameshifting) is important for localization of full-length Ty3 RNA to foci. Ty3 RNA variants associated with PB components via independent determinants in the native Ty3 untranslated regions (UTRs) and in GAG3-POL3 sequences flanked by UTRs adapted from non-Ty3 transcripts. However, despite localization, RNAs containing GAG3-POL3 but lacking Ty3 UTRs were not packaged efficiently. Surprisingly, sequences within Ty3 UTRs, which bind the initiator tRNA(Met) proposed to provide the dimerization interface, were not required for packaging of full-length Ty3 RNA into VLPs. In summary, our results demonstrate that Gag3 is sufficient and required for localization and packaging of RNAs containing Ty3 UTRs and support a role for POL3 sequences, translation of which is attenuated by programmed frameshifting, in both localization and packaging of the Ty3 full-length gRNA.

Figure 1. Ty3 genome and expression variants

A. Full-length Ty3 DNA (U3RU5-coding region-U3RU5) and RNA (RU5-coding region-U3R) sequences. First and second ORFs GAG3 and POL3 are translated into Gag3 and Gag3-Pol3 polyproteins and processed into the mature species indicated. The Ty3 gRNA 5’UTR is comprised of RU5 and 75 nt of the internal domain upstream of the initiator AUG. POL3 extends into the downstream LTR by 46 nts. For simplicity in the text, Ty3 “UTRs” refers to the 5’ UTR and the complete 3’ U3R (of which 46 nts is translated). B. Ty3-related expression constructs. The GAL1-10 UAS (GAL10 orientation) partially replaced U3 in the upstream LTR to allow galactose-regulated expression of Ty3-related transcripts (left-hand column). *Indicates stop codon. Right-hand column displays pYES2.0-based full length Ty3, GAG3 and LacZ reporter expression constructs. pYES2.0-based constructs containing a 5’ UTR derived from Ty3 have the RU5 sequence placed downstream of the GAL1 promoter. hUTRs were derived by transcription from pYES2.0 under control of the GAL1-10 UAS, the GAL1 promoter and 5’ UTR, and the CYC1 terminator. Empty white boxes denote hUTRs. Complete annotated plasmid sequences are presented in Supplemental Materials.

Figure 2. Expression of Ty3-related RNAs and proteins

A. Ty3 Gag3 expression. Total Ty3 Gag3 and Gag3 processing products in WCEs were quantified using a LAS-4000 Fuji imaging system and Multigauge V3.0 analysis software. This level was normalized to the Pgk1 loading control, and these values were normalized to the wt Ty3 protein signal. Two transformants of each sample were quantified and averaged, and a representative blot is shown. α-CA, anti-capsid. α-Pgk1, anti-Pgk1. B. Ty3 RNA levels. Two independent transformants were analyzed by RT-qPCR using pairs of primers that annealed in GAG3 and in ACT1 RNA. RT-qPCR was performed in the linear concentration range for two independent transformants, each in triplicate. Triplicates were averaged and Ty3 GAG3 signals were normalized to those of ACT1. The average normalized GAG3 signal of the two transformants is presented. Ctl, uninduced control. C. RNAs containing Ty3 UTRs have lower Ty3 protein/RNA ratios. The ratio of Ty3 protein/RNA (determined as in A and B) was determined for cells expressing Ty3 RNA variants. UTRs, grey shaded boxes; hUTRs, white boxes.

Figure 3. Ty3 sequences in UTRs and POL3 independently mediate localization to foci with PB reporters

Confocal fluorescence microscopy localization of Ty3-related RNAs. BY4741 expressing the reporter protein Dhh1-GFP or Dcp2-GFP was transformed with Ty3-MS2 and MS2-RFP expression plasmids and induced as described in Methods. Images are representative of two independent transformants of each type. A minimum of 150 MS2-RFP-expressing cells were analyzed for each variant. The galactose-induced Ty3-MS2 RNA is indicated schematically to the left of the fluorescence image (for details see Fig. 1 legend). The top row of panels depict cells that were uninduced, while in the rest of the panels show induced cells. Inset numbers in red indicate number of RFP foci (greater than 0.1µm²)/MS2-RFP-expressing cells. Inset numbers in yellow indicate the percentage of MS2-RFP foci overlapping Dhh1-GFP or Dcp2-GFP reporter foci. The scale bar is 5 µm. Horizontal bars between images: relative size distribution of MS2-RFP foci measured as area of foci (in µm²). Upper bar of pair: Dhh1-GFP; lower bar: Dcp2-GFP. Scale (white bar) is 5 µm. Data processing was as described in Methods.

Figure 4. Packaging of variant Ty3 RNAs into VLPs

Extracts of cells expressing Ty3 RNAs were prepared under non-denaturing conditions after 24 h (A) or 6 h (B and C) of galactose induction. An equal amount of in vitro transcribed, truncated Ty3 RNA (Ty3 runoff RNA) was added to each WCE to monitor nuclease digestion. Nuclease treatment was performed as described in Methods. Northern Blots were probed with a ³²P-labeled DNA fragment specific to Ty3 RNA and quantified with Quantity One Software (Bio-Rad, Hercules, CA). Two or more independent experiments were performed for each Ty3 RNA variant, and a representative example is shown. % protection represents the amount of nuclease-treated RNA divided by the amount of untreated RNA. A. Localization of Ty3 RNA with PB proteins in foci does not insure packaging. B. The 5’ UTR of Ty3 is sufficient to direct packaging of short GAG3-containing RNAs. C. Deletion of the bipartite PBS sequences does not disrupt full-length Ty3 RNA packaging.

Figure 5. Gag3 packages Ty3 UTR-containing RNA in trans

Yeast strain BY4741 containing trp1Δ was transformed with a low-copy vector plasmid carrying lacZ with Ty3-specific UTRs or hUTR; these were co-transformed with low-copy helper plasmid carrying GAG3 with hUTRs and low-copy plasmid carrying MS2-RFP reporter (see Table 1 and Methods). Samples were induced by addition of galactose and allowed to grow for 6 h (see Methods). The scale bar is 5 µm.