Converting nonsense codons into sense codons by targeted pseudouridylation

Abstract

All three translation termination codons, or nonsense codons, contain a uridine residue at the first position of the codon. Here, we demonstrate that pseudouridylation (conversion of uridine into pseudouridine (Ψ), ref. 4) of nonsense codons suppresses translation termination both in vitro and in vivo. In vivo targeting of nonsense codons is accomplished by the expression of an H/ACA RNA capable of directing the isomerization of uridine to Ψ within the nonsense codon. Thus, targeted pseudouridylation represents a novel approach for promoting nonsense suppression in vivo. Remarkably, we also show that pseudouridylated nonsense codons code for amino acids with similar properties. Specifically, ΨAA and ΨAG code for serine and threonine, whereas ΨGA codes for tyrosine and phenylalanine, thus suggesting a new mode of decoding. Our results also suggest that RNA modification, as a naturally occurring mechanism, may offer a new way to expand the genetic code.

Fig. 1

Pseudouridylation of a termination codon promotes nonsense suppression in vitro. (a) Nucleotide sequence of the in vitro transcription product and its translated sequence are shown. Positions of the Kozak sequence, as well as epitopes (6HIS and FLAG) within the nucleotide and protein sequences are labeled. The pseudouridylated nonsense codon is indicated. Changes of Ψ to U and Ψ to C are also indicated. (b) Anti-6HIS and anti-FLAG immunoblot analysis of the in vitro translation lysate following translation of an RNA lacking a termination codon (CAA), an RNA containing a pseudouridylated termination codon (ΨAA), or an RNA containing an authentic termination codon (UAA). Relative efficiency of read-through (anti-FLAG/anti-6HIS) was calculated and indicated in parentheses (the control, CAA, is set to 100%). Error is given as the standard deviation of three independent experiments.

Fig. 2

Quantification of cup1-PTC pseudouridylation. (a) Schematic of the in vivo nonsense suppression assay. (b) In vitro pseudouridylation assay by thin layer chromatography. 5′ ³²P-radiolabeled uridylate (pU) and pseudouridylate (pΨ) markers were run in parallel. The substrate—[α³²P]UTP uniformly labeled RNA fragment—is shown. (c) Quantification of cup1-PTC pseudouridylation in vivo. The percentage of pseudouridylation was calculated [pΨ/(pΨ + pU)]. Adenosine 5′-monophosphate (pA), cytidine 5′-monophosphate (pC), guanosine 5′-monophosphate (pG), uridine 5′-monophosphate (pU), and pseudouridine 5′-monophosphate (pΨ) are indicated. (●) Origin. Error is given as the standard deviation of three independent experiments.

Fig. 3

Expression of an H/ACA RNA targeting the PTC of cup-PTC for pseudouridylation promotes nonsense suppression. (a) pCUP1 or pcup1-PTC along with either an empty vector or psnR81-1C were transformed into a cup1Δ strain. Cell growth was assessed on solid synthetic medium (-URA -LEU) containing either 0.0 mM or 0.02 mM CuSO₄, as indicated. (b) Northern blot analysis of RNA extracted from cells described in (a). Normalized levels of CUP1 mRNA (lane 5) and cup1-PTC mRNA (lanes 6 and 7) are indicated in parentheses under each lane. Error is given as the standard deviation of three independent experiments. (c) cup1Δ upf1Δ strain was transformed with either pCUP1 or pcup1-PTC along with either psnR81-Random or psnR81-1C. Cell growth was assessed on solid synthetic medium (-HIS -URA -LEU) with or without CuSO₄, as indicated. (d) Northern blot analysis of RNA extracted from cells described in (c). Normalized levels of CUP1 mRNA (lane 5) and cup1-PTC mRNA (lanes 6 and 7) are indicated in parentheses under each lane. Error is given as the standard deviation of three independent experiments.

Fig 4

Generalization of Ψ-mediated nonsense suppression and determination of amino acids coded for by pseudouridylated nonsense codons. (a) Schematic representation of the constructs used for protein purification (also see text). (b) Western blot analysis was carried out using extracts prepared from wild-type cells transformed with either pTRM4 WT and a plasmid containing a random guide RNA gene (pRandom guide RNA) (lane 4), pTRM4-F602X(TAA) and pRandom guide RNA (lane 5), or pTRM4-F602X(TAA) and a plasmid containing a guide RNA gene that targets the nonsense codon (UAA 602) of TRM4-F602X(TAA) (lane 6). Enolase was probed as a loading control. The normalized levels of Trm4p are indicated in parentheses under each lane. Error is represented as the standard deviation from three independent experiments. (c) Cell cultures described in (b) were scaled up, and Trm4 proteins were purified and analyzed on a SDS-PAGE gel (stained with coomassie blue); lanes correspond to those in (b). In the control lane (Con), a known amount (6 μg) of purified Trm4p was loaded. (d) Identification of amino acids incorporated at Ψ-containing termination codons (also see Supplementary Figs. 4–8).