Maas Research Group
Department of Biological Sciences, Lehigh University
Overview
Our lab is interested in understanding the molecular basis for the tremendous complexity and diversity of higher organisms. This complexity is based on the number of different gene products available for structural, enzymatic and regulatory functions.
We are focusing on a recently discovered phenomenon of pre-mRNA modification, called RNA editing. In general, the term RNA editing is used to describe the posttranscriptional alteration of gene sequences by different mechanisms including the deletion, insertion and modification of nucleotides. Base changes in codons often lead to amino acid changes and result in alteration of protein function.
In addition to the recoding of mRNA information, the modification of RNAs by ADARs could potentially affect any biological process that involves sequence- or structure-specific interactions with RNA.
We use molecular biological, biochemical and genetics approaches to study how prevalent A-to-I RNA editing is in the transcriptome and how it is regulated. Furthermore, we investigate the consequences of misregulation of or deficiency in RNA editing and how it can contribute to pathophysiological processes.
The complexity of higher organisms is based on the number of different gene products available for structural, enzymatic and regulatory functions. Recently, whole-genome sequencing of a diverse set of species from primitive to highly developed organisms (bacteria, yeast, worm, fly, fish, mouse, human) has revealed that during evolution the number of genes in genomes has not increased at the same rate as the observed organismal complexity (1). For example, the fruit fly (D. melanogaster) carries fewer genes than the nematode C. elegans (~14,000 (2) versus ~19,000 (3)) even though the fly is obviously more complex. And when comparing these numbers from invertebrates to the estimated number of genes in the human genome (ca. 30,000 (4,5)), it is inconceivable that the increase in gene numbers alone could be the basis for the tremendous evolutionary increase in diversity and complexity.
These findings underscore the important role the mechanisms that control gene utilization play in the creation of proteomic and phenotypic diversity. Several post-transcriptional and post-translational mechanisms have been identified that lead to the production of multiple gene products from a single gene. The alternative splicing of pre-mRNAs is a particularly frequent event, estimated to affect more than 50% of mammalian primary transcripts (6).
Another post-transcriptional processing pathway that appears to be widespread in mammals is RNA editing by adenosine (A) deamination, by which individual adenosine bases in pre-mRNA are modified to yield inosine (I). Since inosine acts as guanosine during translation, A-to-I conversion in coding sequences leads to amino acid changes and often entails changes in protein function (7-9). The best studied examples are neuronal glutamate receptor genes where single amino acid changes alter gating behavior and kinetic properties of the ion channels (10-13) and a serotonin receptor subunit where editing regulates G-protein coupling efficiency (14).
The power of RNA editing in the generation of protein diversity lies in the fact that usually both the edited and unedited versions of the protein are co-expressed in the same cell and the ratio between the unedited and multiple edited variants can be regulated in a cell type-specific or time-dependent manner. Furthermore, editing within non-coding regions of pre-mRNAs can lead to alternative splicing or might affect the translation, transport or stability of RNAs. Figure 1 depicts where in primary gene transcripts A-to-I editing has been observed to date with the known or proposed sequels for the modified RNA.
Gene regulation through A-to-I RNA editing |
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A-to-I RNA editing is catalyzed by the ADAR (adenosine deaminase acting on RNA) family of enzymes (8) and orthologues of mammalian ADARs have also been identified in D.melanogaster (15), C. elegans (16) and fish species (17,18). These proteins recognize a double-stranded RNA structure formed from exonic and intronic sequences within the substrate molecule (19).
The modification of RNAs by ADARs could potentially affect any biological process that involves sequence- or structure-specific interactions with RNA. Recent studies indicate that the editing activity of ADARs might also be involved in the antiviral interferon response pathway, where extensive deamination of viral dsRNAs, termed hypermutation (20), could inhibit viral transcription and replication (8,20-23). Interestingly, Hepatitis Delta Virus (HDV) exploits the cellular A-to-I editing machinery to convert of a translational stop signal into a tryptophan codon within its only open reading frame (24). This editing event is essential for the viral life cycle since the unedited and edited protein variants have specific functions in replication and virus assembly (25,26).
Mammalian genes that undergo A-to-I RNA editing
In general, the term RNA editing is used to describe the post-transcriptional alteration of gene sequences by diverse mechanisms including the deletion, insertion and modification of nucleotides (7,27,28). In mammals, two types of RNA editing have been found that involve deamination of either adenosine or cytidine in nuclear-encoded RNAs to yield inosine or uridine, respectively (29). The C-to-U editing of apolipoprotein B mRNA was the first case of mammalian RNA editing discovered (30) and here the specific deamination of U6666 changes a Gln codon (CAA) to a stop codon (UAA). This results in the production of a shorter protein, apoB48, whereas the unedited apolipoprotein B mRNA results in the translation of the full-length protein apoB100. ApoB100 and apoB48 have distinct functions in lipoprotein metabolism and are associated with different susceptibilities to atherosclerosis (28). Only few cases of C-to-U RNA editing have been identified to date, however, a large family of candidate cytidine deaminases exists in mammalian genomes, some of which have been shown to target single stranded DNA (31-33) instead of RNA.
Since the initial discovery of A-to-I mRNA editing in mammals about a decade ago (10), this process of posttranscriptional modification is now recognized as an important mechanism for the generation of molecular diversity. Through the site-specific recoding of mRNA sequences, A-to-I RNA editing regulates important functional properties of neurotransmitter receptors in the central nervous system (11). In particular, the glutamate receptor subunit GluR-B undergoes almost quantitative editing (>99.9 %) at one position (the Q/R-site), which represents the molecular determinant for low Ca2+-permeability of the ion channel (11). In addition, the Q/R-site regulates the intracellular trafficking of the receptor protein (34).
Transgenic mice with even slightly reduced GluR-B editing suffer severe epileptic seizures and die within 2 weeks of age (35). This phenotype was a consequence of the increased Ca2+-permeability of the underedited glutamate receptors (35). The same phenotype resulted when the editing enzyme ADAR2 was inactivated in mice, due to a dramatic reduction in Q/R-site editing (36).
Other neuronal genes affected by A-to-I RNA editing include the glutamate receptor subunits GluR-C, -D, -5, and -6 where RNA editing regulates gating and kinetic properties of the ion channels (12,13), and the 5HT2C serotonin receptor where editing is known to regulate the receptor’s G-protein coupling efficiency (14). The cell-type specific and developmental regulation of editing-levels at multiple nucleotides in these mRNAs leads to the production of an array of receptor subtypes that differ in one or several amino acids from the unedited counterparts (9,14,29).
In addition to these cases where the editing occurs in coding regions of gene transcripts, A-to-I editing events have also been detected in noncoding regions of cellular genes (37,38) and within viral RNAs after infection (20,24). In one case, the editing enzyme ADAR2 edits its own pre-mRNA creating a new splice site that leads to alternative splicing of the pre-mRNA (38). The editing events seen in 5’- and 3’- untranslated regions of gene transcripts, as well as the ones affecting intronic sequences, might point toward yet unexplored functional roles of editing in the regulation of transport, stability, and further processing of RNAs.
Molecular Mechanism and substrate requirements for site-selective RNA editing
Mechanistically, the A-to-I editing reaction follows a hydrolytic deamination mechanism catalyzed by a Zn2+ coordinating enzyme, which likely is active as a dimer and does not require accessory factors for activity (8). The ADARs are distantly related to DNA methyl-transferases (39) and believed to employ a similar base-flipping mechanism for base modification (8). Double-stranded RNA binding domains and the catalytic region of the RNA editing enzyme convey dsRNA specific adenosine deaminase activity, which on extended perfect dsRNA proceeds promiscuously. The high site-selectivity and specificity of the RNA editing reactions involving physiological substrates is achieved in large part by the overall three dimensional structure of the imperfectly base-paired pre-mRNA with bulges and loops. However, the basis for the selectivity observed for certain RNAs is poorly understood as is the interplay between catalytic and RNA-binding domains of the editing enzymes.
To date it is not possible to predict which adenosine within a given RNA will undergo editing, or if a computer-predicted RNA secondary structure will be a competent in vivo substrate for an ADAR enzyme. When analyzing the predicted RNA secondary structures of endogenous transcripts known to be subject to A-to-I editing, the modified adenosines reside in base-paired regions, mismatched or within a loop (40). Minigene substrates and compensatory mutational analysis have been used to confirm the predicted RNA folds (12,19), however, the three-dimensional structures and interactions of RNA and protein components are not known. Through in vitro studies and analysis of the modification patterns of endogenous substrates a 5’ neighbor preference (ADAR1, 2) and 3’ neighbor preference (ADAR2) have been established (41,42).
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