An evaluation of detection methods for large lariat RNAs

Abstract

Ty1 elements are long terminal repeat (LTR) retrotransposons that reside within the genome of Saccharomyces cerevisiae. It has been known for many years that the 2'-5' phosphodiesterase Dbr1p, which debranches intron lariats, is required for efficient Ty1 transposition. A recent report suggested the intriguing possibility that Ty1 RNA forms a lariat as a transposition intermediate. We set out to further investigate the nature of the proposed Ty1 lariat branchpoint. However, using a wide range of techniques we were unable to find any evidence for the proposed lariat structure. Furthermore, we demonstrate that some of the techniques used in the initial study describing the lariat are capable of incorrectly reporting a lariat structure. Thus, the role of the Dbr1 protein in Ty1 retrotransposition remains elusive.

FIGURE 1.

Electrophoretic mobility of in vitro circularized Ty1 RNA. (A) Schematic representation of the short (Ty1 nucleotides 241–478 adjacent to 3946–5463; Boeke et al. 1988) and long (Ty1 nucleotides 241–5463; Boeke et al. 1988) SP6 promoter driven Ty1 RNA in vitro transcription constructs. The positions of PvuII (P) and SnaBI (S) restriction sites, used respectively to create the deletion for the short Ty1 construct and to linearize the template prior to transcription are shown. The position of the Ty1-specific riboprobe (Ty1 nucleotides 4232–4356; Boeke et al. 1988) used in hybridizations is indicated by a solid box. (B) Northern blot to confirm circularization of short Ty1 construct. Transcribed short RNAs were in vitro ligated using T4 DNA ligase, dephosphorylated with calf alkaline phosphatase, and then labeled with either γ³²P-ATP or ATP using T4 polynucleotide kinase. Following electrophoresis through denaturing polyacrylamide (2.5%/8 M urea), gel sections were exposed directly to X-ray film (γ-³²P labeled) or electroblotted to nylon and probed with the Ty1-specific riboprobe. (C) Northern blot analysis of circularized long Ty1 RNA construct. Transcribed long Ty1 RNAs were in vitro ligated using T4 DNA ligase. Ligated RNAs were separated through 1.3% agarose formaldehyde-containing gels alongside 1 μg VLP and 10 μg total RNA from dbr1 mutant yeast. Northern blots were probed with the Ty1-specific riboprobe. The positions of Ambion Millennium Marker RNA molecular weight standards are indicated. (D) Experiment identical to C except that samples were separated through denaturing polyacrylamide (2.5%/8 M urea). (E) Formaldehyde agarose gel electrophoresis of 100 ng VLP and 5 μg total RNA from independent preparations of dbr1 mutant yeast cell RNA, separated and probed as in C. The positions of Ambion Millennium Marker RNA standards are indicated.

FIGURE 2.

Northern analysis of RNase H-digested Ty1 RNA. (A) Targeted RNase H digestion of Ty1 RNA with two DNA oligonucleotides. Schematics indicate the annealing position of targeting (C,D) and probe (1) oligonucleotides, and the predicted size of RNase H digestion fragments, excluding the size of the polyA tail expected to be present on the lariat Ty1 digestion fragment. RNAs were digested with increasing quantities of oligonucleotide: 0 ng, 1 ng, 2.5 ng, 5 ng, and 10 ng as indicated. Arrows indicate the positions of major RNase H digestion fragments. (B) Targeted RNase H digestion of Ty1 RNA with a single oligonucleotide. Schematics indicate the positions of single targeting (A–C) and probe (1 and 2) oligonucleotides used and the predicted sizes of RNase H digestion fragments, excluding the size of the polyA tail. RNAs were digested with 50 ng of the oligonucleotide indicated. The positions of major digestion fragments are indicated by arrows. It is important to note that the fragments labeled 839 nt and 941 nt do not include a size adjustment for the polyA tail. RNase H digestions shown in both A and B were separated on the same gel, and the Northern blot sequentially hybridized with probes 1 and 2.

FIGURE 3.

(A) Primer extension analysis of Ty1 RNA. Schematic indicates the annealing position and orientation of primers (6673, 6715, and 6716) on both lariat and linear RNA templates. Samples numbered 1 and 3 represent two independent preparations of wild-type VLP RNA; samples numbered 2 and 4 represent two independent preparations of dbr1 mutant VLP RNA. RNA samples 1 and 2 were prepared from cells induced for 24 h, and samples 3 and 4 were from cells induced for 6 h. Samples numbered 5 and 6 are no RNA and no AMV RT negative controls, respectively. The positions of molecular weight markers are indicated. The arrowhead indicates the position of the positive control 149-nt primer extension product. (B) RLM-RACE analysis of RNA prepared from both wild-type and dbr1 mutant yeast strains expressing either a wild-type or RT mutant GAL-Ty1 construct. Reactions were performed both with and without pretreatment of the RNA with phosphatase and pyrophosphatase (Phos +/−). RLM-RACE RT-PCR was performed using a sense primer specific to the 5′ annealed RNA oligo and a gene-specific antisense primer. RACE products were cloned and sequenced for verification. As an internal control for reverse transcription and Ty1 RNA concentration, RT-PCR was performed using the same reactions with internal primers 6668 and 6669. (C) Semiquantitative RLM-RACE RT-PCR was performed exactly as described in B. The number of cycles for which RT-PCR was performed is indicated. (D) RT-PCR across the lariat branch-point. Ty1 RNA prepared from both wild-type and dbr1 mutant VLPs was subjected to RT-PCR using primers 6673 and 6675 spanning the putative branchpoint. The number of PCR cycles performed is indicated. RT-PCR products were cloned and sequenced for product verification. (E) Generation of RT-PCR products from lariat or linear template RNA. An RT-PCR product could be generated by crossover PCR: 1) cDNA synthesis; 2) first-round PCR—annealing of the sense primer followed by extension; 3) second-round PCR—annealing of the first-round PCR product to the opposite end of the cDNA molecule due to complementarity between LTR regions (arrowheads), followed by extension; 4) third-round PCR—annealing of the antisense primer and extension to generate double-stranded PCR product. Similar crossover reaction products can in principle be obtained even in the absence of a full-length cDNA as illustrated in step 1. (F) Assessment of crossover PCR. RT-PCR was performed using in vitro transcribed full-length (FL; Ty1 nucleotides 241–5918) or 3′-truncated (5′ only; Ty1 nucleotides 241–5463) Ty1 RNAs, exactly as described in D.

FIGURE 4.

Ribonuclease protection assays. (A) RNase T1 protection of Ty1 RNA from wild-type and dbr1 mutant cells. Schematics indicate the positions of the unique Ty1 RNA LTR sequences U5 and U3, and the repeated LTR R region (the direction of the R repeat is indicated by arrows). The predicted sizes of RNase protection fragments are shown. RNase protection reaction conditions are indicated above each lane. RNAs were prepared from either wild-type (WT) or dbr1 mutant strains expressing either wild-type (WT, lanes 3,4) or reverse transcriptase mutant (RT-, lanes 5–8) GAL-Ty1 expression plasmids. Ten percent of the no RNase T1 control was loaded in lane 1. To confirm complete digestion of the probe by RNase T1, and to control against false positive results due to probe self-protection, mouse liver RNA (M) was used as a nonspecific RNA target in lane 2. An in vitro transcribed sense RNA (corresponding to Ty1 nucleotides 5413–5824 joined to 241–486) was used as a positive control for protection by the putative lariat RNA structure (lane 9). The sequence of the RNA probe complementary to Ty1 corresponds to nucleotides 389–241 adjacent to 5824–5475. Sizes of RNase protection fragments detected are indicated. (B) Model for RNA probe protection by covalent and non-covalent “pseudolariats.” The positions of G residues (substrate for RNase T1 digestion) in the probe nearest to the proposed branch are indicated in bold type. A true covalent lariat structure of the type proposed would be predicted to protect a full-length complementary probe independent of template RNA concentration. At high template concentration (such as when template is present in molar excess of probe), the probe is predicted to artificially create a non-covalent pseudolariat structure resulting in detection of a full-length protection fragment. At low template concentration (when probe is present in molar excess of template), each linear template RNA molecule would likely have a probe hybridized at each end, preventing artificial lariat detection. (C) Titration of input RNAs. RNase protection was performed exactly as in A, except that the quantity of input template RNA was titrated over a single order of magnitude (VLP RNA dilutions: 1, 1/4, 1/10; total RNA dilutions: 1, 1/2.5, 1/10; in vitro transcribed RNA dilutions: 1, 1/25, 1/100). A larger linear probe fragment (450 nt) is protected by in vitro transcribed template (Ty1 nucleotides 241–5918), since the transcribed RNA includes the U3 region at the 3′ end of the RNA that is not normally present in endogenous Ty1 RNA samples. (D) Template VLP or total RNA was pre-incubated with of 0, 40, 120, or 240 ng purified Dbr1p (Nam and Boeke 1994) and then subjected to RNase protection exactly as described in C. (E) Verification of debranching by Dbr1p. An aliquot of each of the total RNAs used for RPA was separated on a 5% polyacrylamide/8 M urea gel, electroblotted to nylon membrane, and hybridized with an actin intron-specific probe. The position of branched and debranched intron lariat species are indicated.