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One RNA plays three roles to provide catalytic activity to a group I intron lacking an endogenous internal guide sequence

Vaidya N, Lehman N - Nucleic Acids Res. (2009)

Bottom Line: Such multifunctionality would be particularly significant if the phenotypes were functionally inter-related in a common biochemical pathway.Analysis of these reactions indicates that a single RNA fragment can perform up to three distinct roles in a reaction: behaving as a portion of a catalyst, behaving as a substrate, and providing an exogenous IGS.This property of RNA to be multifunctional in a single reaction pathway bolsters the probability that a system of self-replicating molecules could have existed in an RNA world during the origins of life on the Earth.

Affiliation: Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207, USA.

ABSTRACT

Catalytic RNA molecules possess simultaneously a genotype and a phenotype. However, a single RNA genotype has the potential to adopt two or perhaps more distinct phenotypes as a result of differential folding and/or catalytic activity. Such multifunctionality would be particularly significant if the phenotypes were functionally inter-related in a common biochemical pathway. Here, this phenomenon is demonstrated by the ability of the Azoarcus group I ribozyme to function when its canonical internal guide sequence (GUG) has been removed from the 5' end of the molecule, and added back exogenously in trans. The presence of GUG triplets in non-covalent fragments of the ribozyme allow trans-splicing to occur in both a reverse splicing assay and a covalent self-assembly assay in which the internal guide sequence (IGS)-less ribozyme can put itself together from two of its component pieces. Analysis of these reactions indicates that a single RNA fragment can perform up to three distinct roles in a reaction: behaving as a portion of a catalyst, behaving as a substrate, and providing an exogenous IGS. This property of RNA to be multifunctional in a single reaction pathway bolsters the probability that a system of self-replicating molecules could have existed in an RNA world during the origins of life on the Earth.

Schematic of the partitioning of the Azoarcus ribozyme into fragments. (A) The 197-nt source molecule was partitioned into five fragments (U, gray; V, red; X; yellow; Y, blue; Z, green) such that catalytic activity in trans, plus covalent self-assembly could be assayed. Removal of the U fragment from the system at the location indicated by the arrow leaves an L–30 construct which can then be divided into four fragments that can exhibit activity when 5-nt head groups (h = GGCAU) are appended to the 5′ portions of X, Y and Z (see text). A substrate oligomer (lower-case letters = head; black line = tail) binds via a 5′-CAU-3′ to the IGS (GUG) of the ribozyme, which catalyzes phosphotransfer of the tail to its own 3′ end. The native IGS of the ribozyme is denoted by GUG1, while the four exogenous GUG triplets that occur naturally in the remainder of the ribozyme are gray boxes denoted 2–5 (in circles). The tertiary interactions that hold the IGS and the IGS complement into the active site as determined by X-ray crystallography (24) are denoted using the hydrogen-bonding symbolism of Leontis et al. (41). (B) Schematic of how a ribozyme complex (either as a covalently contiguous molecule or as several fragments cooperating in trans) can perform catalysis in the absence of the U fragment that contains the native IGS. Here, the use of an exogenous IGS (GUG5) present in the h•Z fragment to bind to the IGS complement CAU is depicted.
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Figure 1: Schematic of the partitioning of the Azoarcus ribozyme into fragments. (A) The 197-nt source molecule was partitioned into five fragments (U, gray; V, red; X; yellow; Y, blue; Z, green) such that catalytic activity in trans, plus covalent self-assembly could be assayed. Removal of the U fragment from the system at the location indicated by the arrow leaves an L–30 construct which can then be divided into four fragments that can exhibit activity when 5-nt head groups (h = GGCAU) are appended to the 5′ portions of X, Y and Z (see text). A substrate oligomer (lower-case letters = head; black line = tail) binds via a 5′-CAU-3′ to the IGS (GUG) of the ribozyme, which catalyzes phosphotransfer of the tail to its own 3′ end. The native IGS of the ribozyme is denoted by GUG1, while the four exogenous GUG triplets that occur naturally in the remainder of the ribozyme are gray boxes denoted 2–5 (in circles). The tertiary interactions that hold the IGS and the IGS complement into the active site as determined by X-ray crystallography (24) are denoted using the hydrogen-bonding symbolism of Leontis et al. (41). (B) Schematic of how a ribozyme complex (either as a covalently contiguous molecule or as several fragments cooperating in trans) can perform catalysis in the absence of the U fragment that contains the native IGS. Here, the use of an exogenous IGS (GUG5) present in the h•Z fragment to bind to the IGS complement CAU is depicted.

Mentions: Group I introns catalyze RNA phosphoester transfer reactions at specific splice sites both in vivo and in vitro. The splice site selection is precise, and relies on base-pairing interactions between the 5′ portion in P1 of the catalytic intron and a pseudo-complementary 3–6-nt region of the 3′ portion of the 5′ exon (1,2). The former has been termed the internal guide sequence (IGS) and the latter can be referred to as the IGS complement. Base-pairing between the IGS and its complement depends on Watson–Crick pairing at most positions; however the 3′ nt of the IGS complement always forms a G•U wobble pair with the IGS to define precisely the 5′ splice site (1–4). For example, the IGS of the group I intron in the tRNAIle transcript from the purple bacterium Azoarcus can be shortened in vitro to 5′-GUG-3′, and this pairs with the complement 5′-CAU-3′ to effect splicing after the terminal U in the complement (5–7) (Figure 1A).Figure 1.

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One RNA plays three roles to provide catalytic activity to a group I intron lacking an endogenous internal guide sequence

Vaidya N, Lehman N - Nucleic Acids Res. (2009)

Schematic of the partitioning of the Azoarcus ribozyme into fragments. (A) The 197-nt source molecule was partitioned into five fragments (U, gray; V, red; X; yellow; Y, blue; Z, green) such that catalytic activity in trans, plus covalent self-assembly could be assayed. Removal of the U fragment from the system at the location indicated by the arrow leaves an L–30 construct which can then be divided into four fragments that can exhibit activity when 5-nt head groups (h = GGCAU) are appended to the 5′ portions of X, Y and Z (see text). A substrate oligomer (lower-case letters = head; black line = tail) binds via a 5′-CAU-3′ to the IGS (GUG) of the ribozyme, which catalyzes phosphotransfer of the tail to its own 3′ end. The native IGS of the ribozyme is denoted by GUG1, while the four exogenous GUG triplets that occur naturally in the remainder of the ribozyme are gray boxes denoted 2–5 (in circles). The tertiary interactions that hold the IGS and the IGS complement into the active site as determined by X-ray crystallography (24) are denoted using the hydrogen-bonding symbolism of Leontis et al. (41). (B) Schematic of how a ribozyme complex (either as a covalently contiguous molecule or as several fragments cooperating in trans) can perform catalysis in the absence of the U fragment that contains the native IGS. Here, the use of an exogenous IGS (GUG5) present in the h•Z fragment to bind to the IGS complement CAU is depicted.
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Figure 1: Schematic of the partitioning of the Azoarcus ribozyme into fragments. (A) The 197-nt source molecule was partitioned into five fragments (U, gray; V, red; X; yellow; Y, blue; Z, green) such that catalytic activity in trans, plus covalent self-assembly could be assayed. Removal of the U fragment from the system at the location indicated by the arrow leaves an L–30 construct which can then be divided into four fragments that can exhibit activity when 5-nt head groups (h = GGCAU) are appended to the 5′ portions of X, Y and Z (see text). A substrate oligomer (lower-case letters = head; black line = tail) binds via a 5′-CAU-3′ to the IGS (GUG) of the ribozyme, which catalyzes phosphotransfer of the tail to its own 3′ end. The native IGS of the ribozyme is denoted by GUG1, while the four exogenous GUG triplets that occur naturally in the remainder of the ribozyme are gray boxes denoted 2–5 (in circles). The tertiary interactions that hold the IGS and the IGS complement into the active site as determined by X-ray crystallography (24) are denoted using the hydrogen-bonding symbolism of Leontis et al. (41). (B) Schematic of how a ribozyme complex (either as a covalently contiguous molecule or as several fragments cooperating in trans) can perform catalysis in the absence of the U fragment that contains the native IGS. Here, the use of an exogenous IGS (GUG5) present in the h•Z fragment to bind to the IGS complement CAU is depicted.
Mentions: Group I introns catalyze RNA phosphoester transfer reactions at specific splice sites both in vivo and in vitro. The splice site selection is precise, and relies on base-pairing interactions between the 5′ portion in P1 of the catalytic intron and a pseudo-complementary 3–6-nt region of the 3′ portion of the 5′ exon (1,2). The former has been termed the internal guide sequence (IGS) and the latter can be referred to as the IGS complement. Base-pairing between the IGS and its complement depends on Watson–Crick pairing at most positions; however the 3′ nt of the IGS complement always forms a G•U wobble pair with the IGS to define precisely the 5′ splice site (1–4). For example, the IGS of the group I intron in the tRNAIle transcript from the purple bacterium Azoarcus can be shortened in vitro to 5′-GUG-3′, and this pairs with the complement 5′-CAU-3′ to effect splicing after the terminal U in the complement (5–7) (Figure 1A).Figure 1.

Bottom Line: Such multifunctionality would be particularly significant if the phenotypes were functionally inter-related in a common biochemical pathway.Analysis of these reactions indicates that a single RNA fragment can perform up to three distinct roles in a reaction: behaving as a portion of a catalyst, behaving as a substrate, and providing an exogenous IGS.This property of RNA to be multifunctional in a single reaction pathway bolsters the probability that a system of self-replicating molecules could have existed in an RNA world during the origins of life on the Earth.

Affiliation: Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207, USA.

ABSTRACT

Catalytic RNA molecules possess simultaneously a genotype and a phenotype. However, a single RNA genotype has the potential to adopt two or perhaps more distinct phenotypes as a result of differential folding and/or catalytic activity. Such multifunctionality would be particularly significant if the phenotypes were functionally inter-related in a common biochemical pathway. Here, this phenomenon is demonstrated by the ability of the Azoarcus group I ribozyme to function when its canonical internal guide sequence (GUG) has been removed from the 5' end of the molecule, and added back exogenously in trans. The presence of GUG triplets in non-covalent fragments of the ribozyme allow trans-splicing to occur in both a reverse splicing assay and a covalent self-assembly assay in which the internal guide sequence (IGS)-less ribozyme can put itself together from two of its component pieces. Analysis of these reactions indicates that a single RNA fragment can perform up to three distinct roles in a reaction: behaving as a portion of a catalyst, behaving as a substrate, and providing an exogenous IGS. This property of RNA to be multifunctional in a single reaction pathway bolsters the probability that a system of self-replicating molecules could have existed in an RNA world during the origins of life on the Earth.

View Similar Images In: Results  - Collection
View Article: MedlinePlus - PubMed Central -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=2709566&rFormat=json&query=the&fields=all&favor=none&it=none&sp=none&sub=none&uniq=0&req=5