The Lariat capping ribozyme (formerly called GIR1 branching ribozyme) is a ~180 nt ribozyme with an apparent resemblance to a group I ribozyme. [1] It is found within a complex type of group I introns also termed twin-ribozyme introns. [2] Rather than splicing, it catalyses a branching reaction in which the 2'OH of an internal residue is involved in a nucleophilic attack at a nearby phosphodiester bond. [3] As a result, the RNA is cleaved at an internal processing site (IPS), leaving a 3'OH and a downstream product with a 3 nt lariat at its 5' end. The lariat has the first and the third nucleotide joined by a 2',5' phosphodiester bond and is referred to as 'the lariat cap' because it caps an intron-encoded mRNA. The resulting lariat cap seems to contribute by increasing the half-life of the HE mRNA, [3] [4] thus conferring an evolutionary advantage to the HE.
The GIR1 ribozyme was originally discovered during the functional characterization of the introns from the extrachromosomal rDNA of the Didymium iridis protist. A combination of deletion and in vitro self-splicing analyses revealed a twin-ribozyme intron organization: two distinct ribozyme domains within the intron. [2]
The twin-ribozyme introns represent some of the most complex organized group I introns known and consist of a homing endonuclease gene (HEG: I-DirI homing endonuclease) embedded in two functionally distinct catalytic RNA domains. One of the catalytic RNAs is a conventional group I intron ribozyme (GIR2) responsible for the intron splicing and reverse splicing, as well as intron RNA circularization. The other catalytic RNA domain is the group I-like ribozyme (GIR1) directly involved in homing endonuclease mRNA maturation.
| GIR1 Branching Ribozyme | |
|---|---|
 Conserved secondary structure of GIR1 | |
| Identifiers | |
| Symbol | GIR1 |
| Rfam | RF01807 |
| Other data | |
| RNA type | Intron |
| Domain(s) | Naegleria |
| PDB structures | PDBe |
In vitro, DiGIR1 catalyses three different reactions. The first one consists in hydrolysis of the scissile phosphate at the IPS site. This is the cleavage reaction observed with the full-length intron and several length variants with a relative low rate. The hydrolytic cleavage is irreversible and is considered an in vitro artefact resulting from misfolding of the catalytic site to present the branch nucleotide (BP) correctly for the reaction. The second reaction, the natural one, is the branching reaction, in which a transesterification at the IPS site results in the cleavage of the RNA with a 3'OH and a downstream lariat cap made by joining of the first and the third nucleotide by a 2'-5' phosphodiester bond. [3]
These products are the only products observed by analysis of cellular RNA. [4] [5] This branching reaction is in equilibrium with a third one: a ligation reaction. It is a very efficient reaction and it tends to mask the branching reaction during the in vitro branching experiments with the full-length intron and length variants that include more than 166 nucleotides upstream of the IPS.
GIR1 models have been created using biochemical and mutational data. [6] The structure contains an extended substrate domain which contains a GoU pair. The pair differs from the typical group 1 ribozyme nucleophilic residue, the J8/7 region has been reduced. [6] These findings provide the basis for an evolutionary mechanism that accounts for the change from group I splicing ribozyme to the branching GIR1 architecture. This mechanism could potentially be applied to other large RNAs such as the ribonuclease P. [6]
The crystal structure of the LC ribozyme was recently published. [7] In brief, a circularly permutated (CP) ribozyme RNA was generated by in vitro transcription using T7 RNA polymerase. [8] The 5' and 3' generated by circular permutation are located at the natural ribozyme cleavage site. To allow transcription of this construct, optimized 5' hammerhead and HdV (Hepatitis delay Virus) ribozymes were flanked to the LC CP construct. [9]
The crystal structure of the LC ribozyme unravels how the regulatory domain formed by P2, P2.1 and P10 works. Two sets of tertiary interactions take place to constrain P2 and P2.1 allowing the formation of a 3-way junction, which acts as a receptor for nt A209. This snug fit interaction promotes formation of the catalytic site, provided that the lariat is pre-folded by the ribozyme core.
RNA splicing is a process in molecular biology where a newly-made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). It works by removing all the introns and splicing back together exons. For nuclear-encoded genes, splicing occurs in the nucleus either during or immediately after transcription. For those eukaryotic genes that contain introns, splicing is usually needed to create an mRNA molecule that can be translated into protein. For many eukaryotic introns, splicing occurs in a series of reactions which are catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs). There exist self-splicing introns, that is, ribozymes that can catalyze their own excision from their parent RNA molecule. The process of transcription, splicing and translation is called gene expression, the central dogma of molecular biology.
Ribozymes are RNA molecules that have the ability to catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes. The 1982 discovery of ribozymes demonstrated that RNA can be both genetic material and a biological catalyst, and contributed to the RNA world hypothesis, which suggests that RNA may have been important in the evolution of prebiotic self-replicating systems.
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 5′ to 3′ exonuclease (Xrn1), which is a dependent decapping protein; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease.
Deoxyribozymes, also called DNA enzymes, DNAzymes, or catalytic DNA, are DNA oligonucleotides that are capable of performing a specific chemical reaction, often but not always catalytic. This is similar to the action of other biological enzymes, such as proteins or ribozymes . However, in contrast to the abundance of protein enzymes in biological systems and the discovery of biological ribozymes in the 1980s, there is only little evidence for naturally occurring deoxyribozymes. Deoxyribozymes should not be confused with DNA aptamers which are oligonucleotides that selectively bind a target ligand, but do not catalyze a subsequent chemical reaction.
Transcriptional modification or co-transcriptional modification is a set of biological processes common to most eukaryotic cells by which an RNA primary transcript is chemically altered following transcription from a gene to produce a mature, functional RNA molecule that can then leave the nucleus and perform any of a variety of different functions in the cell. There are many types of post-transcriptional modifications achieved through a diverse class of molecular mechanisms.
Small nuclear RNA (snRNA) is a class of small RNA molecules that are found within the splicing speckles and Cajal bodies of the cell nucleus in eukaryotic cells. The length of an average snRNA is approximately 150 nucleotides. They are transcribed by either RNA polymerase II or RNA polymerase III. Their primary function is in the processing of pre-messenger RNA (hnRNA) in the nucleus. They have also been shown to aid in the regulation of transcription factors or RNA polymerase II, and maintaining the telomeres.
Ribonuclease P is a type of ribonuclease which cleaves RNA. RNase P is unique from other RNases in that it is a ribozyme – a ribonucleic acid that acts as a catalyst in the same way that a protein-based enzyme would. Its function is to cleave off an extra, or precursor, sequence of RNA on tRNA molecules. Further, RNase P is one of two known multiple turnover ribozymes in nature, the discovery of which earned Sidney Altman and Thomas Cech the Nobel Prize in Chemistry in 1989: in the 1970s, Altman discovered the existence of precursor tRNA with flanking sequences and was the first to characterize RNase P and its activity in processing of the 5' leader sequence of precursor tRNA. Recent findings also reveal that RNase P has a new function. It has been shown that human nuclear RNase P is required for the normal and efficient transcription of various small noncoding RNAs, such as tRNA, 5S rRNA, SRP RNA and U6 snRNA genes, which are transcribed by RNA polymerase III, one of three major nuclear RNA polymerases in human cells.
In molecular biology, a twintron is an intron-within-intron excised by sequential splicing reactions. A twintron is presumably formed by the insertion of a mobile intron into an existing intron.
Group II introns are a large class of self-catalytic ribozymes and mobile genetic elements found within the genes of all three domains of life. Ribozyme activity can occur under high-salt conditions in vitro. However, assistance from proteins is required for in vivo splicing. In contrast to group I introns, intron excision occurs in the absence of GTP and involves the formation of a lariat, with an A-residue branchpoint strongly resembling that found in lariats formed during splicing of nuclear pre-mRNA. It is hypothesized that pre-mRNA splicing may have evolved from group II introns, due to the similar catalytic mechanism as well as the structural similarity of the Group II Domain V substructure to the U6/U2 extended snRNA. Finally, their ability to site-specifically insert into DNA sites has been exploited as a tool for biotechnology. For example, group II introns can be modified to make site-specific genome insertions and deliver cargo DNA such as reporter genes or lox sites
I-CreI is a homing endonuclease whose gene was first discovered in the chloroplast genome of Chlamydomonas reinhardtii, a species of unicellular green algae. It is named for the facts that: it resides in an Intron; it was isolated from Clamydomonas reinhardtii; it was the first (I) such gene isolated from C. reinhardtii. Its gene resides in a group I intron in the 23S ribosomal RNA gene of the C. reinhardtii chloroplast, and I-CreI is only expressed when its mRNA is spliced from the primary transcript of the 23S gene. I-CreI enzyme, which functions as a homodimer, recognizes a 22-nucleotide sequence of duplex DNA and cleaves one phosphodiester bond on each strand at specific positions. I-CreI is a member of the LAGLIDADG family of homing endonucleases, all of which have a conserved LAGLIDADG amino acid motif that contributes to their associative domains and active sites. When the I-CreI-containing intron encounters a 23S allele lacking the intron, I-CreI enzyme "homes" in on the "intron-minus" allele of 23S and effects its parent intron's insertion into the intron-minus allele. Introns with this behavior are called mobile introns. Because I-CreI provides for its own propagation while conferring no benefit on its host, it is an example of selfish DNA.
The homing endonucleases are a collection of endonucleases encoded either as freestanding genes within introns, as fusions with host proteins, or as self-splicing inteins. They catalyze the hydrolysis of genomic DNA within the cells that synthesize them, but do so at very few, or even singular, locations. Repair of the hydrolyzed DNA by the host cell frequently results in the gene encoding the homing endonuclease having been copied into the cleavage site, hence the term 'homing' to describe the movement of these genes. Homing endonucleases can thereby transmit their genes horizontally within a host population, increasing their allele frequency at greater than Mendelian rates.
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Numerous key discoveries in biology have emerged from studies of RNA, including seminal work in the fields of biochemistry, genetics, microbiology, molecular biology, molecular evolution, and structural biology. As of 2010, 30 scientists have been awarded Nobel Prizes for experimental work that includes studies of RNA. Specific discoveries of high biological significance are discussed in this article.
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RNA hydrolysis is a reaction in which a phosphodiester bond in the sugar-phosphate backbone of RNA is broken, cleaving the RNA molecule. RNA is susceptible to this base-catalyzed hydrolysis because the ribose sugar in RNA has a hydroxyl group at the 2’ position. This feature makes RNA chemically unstable compared to DNA, which does not have this 2’ -OH group and thus is not susceptible to base-catalyzed hydrolysis.
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