RNA Editing in African Trypanosomes

BioBio Faculty: "The central dogma of Molecular Biology states a linear flow of genetic information: DNA to/from RNA to protein. However, recently discovered processes of RNA editing which modify the genetic material during or after transcription expand this paradigm. A dramatic example is the post-transcriptional maturation of mitochondrial mRNAs by the addition and deletion of U residues in trypanosomes, whereby amino acid coding triplets and stop codons are created. Extensive editing in some mRNAs generates over half their mature size, yet a single error in the U-changes yields a frameshift. Trypanosome RNA editing is developmentally regulated and appears to control mitochondrial respiration.

In vitro studies have revealed that high fidelity RNA editing is directed by small transacting guide RNAs (gRNAs), and is catalyzed by a protein complex or editosome containing endonuclease, exonuclease, terminal transferase and RNA ligase. "

ribozyme group I intron

Making sense of gene therapy: "He uses an RNA enzyme (ribozyme), called a Group I intron, to repair RNA. These introns cleave RNA at two sites, discarding the intervening RNA, before joining the ends back together.

Crucially, if the ribozyme has a sequence attached to its tail end, it sticks this into the RNA gap before gluing the whole thing back together; replacing one piece of RNA with another. In addition, the ribozyme recognizes its cleavage sites by base-pairing, so by changing the ribozyme sequence, it can be engineered to cut at different targets."

mRNA Cap Formation

UB Department of Biological Sciences: Kiong Ho: "Cap formation is mediated by three enzymatic reactions in which the 5' triphosphate end of pre-mRNA is hydrolyzed to a 5' diphosphate by RNA triphosphatase, then capped with GMP by RNA guanylyltransferase, and methylated by RNA (guanine-N7) methyltransferase. The sequential steps in the capping reaction are universal to all eukaryotes, yet there is a significant divergence in the catalytic mechanism of the triphosphatase component. Metazoan triphosphatases belong to a superfamily of phosphatases that act via the formation and hydrolysis of a cysteinyl-phosphate intermediate. Fungal and viral triphosphatases comprise a novel family of metal-dependent phosphohydrolases with a unique tertiary structure."

mRNA Cap Formation in Parasitic Protozoa

UB Department of Biological Sciences: Kiong Ho: "mRNA processing plays a critical role in the expression of eukaryotic genes. Processing occurs cotranscriptionally on nascent chains synthesized by RNA polymerase II. The earliest modification event is the addition of m7GpppN cap. This structural hallmark is present on all eukaryotic cellular mRNAs and is essential for viability. The cap enhances several downstream events in gene expression including mRNA stability, splicing of pre-mRNAs, and initiation of protein synthesis.

Structure of Mammalian RNA Triphosphatase

Structure of Fungal RNA Triphosphatase
Cap formation is mediated by three enzymatic reactions in which the 5' triphosphate end of pre-mRNA is hydrolyzed to a 5' diphosphate by RNA triphosphatase, then capped with GMP by RNA guanylyltransferase, and methylated by RNA (guanine-N7) methyltransferase. The sequential steps in the capping reaction are universal to all eukaryotes, yet there is a significant divergence in the catalytic mechanism of the triphosphatase component. Metazoan triphosphatases belong to a superfamily of phosphatases that act via the formation and hydrolysis of a cysteinyl-phosphate intermediate. Fungal and viral triphosphatases comprise a novel family of metal-dependent phosphohydrolases with a unique tertiary structure. "

RNA modulation, repair and remodeling by splice switching oligonucleotides

"Targeting splicing by antisense oligonucleotides allows RNA modifications that
are not possible with RNA interference or other antisense techniques that destine
the RNA for destruction. By changing the ratio of naturally occurring splice variants
the expression of mRNA is modulated. By preventing the use of an aberrant splice
site created by a mutation and enforcing re-selection of correct splice sites the RNA
is repaired. Antisense induced skipping of the exon that carries a nonsense mutation
remodels the mRNA and restores the reading frame of the defective protein. All of
the above approaches have clinical applications. Modulation of splice variants is particularly
important since close to 60% of all genes code for alternatively spliced pre-mRNA."

Ryszard Kole, Tiffany Williams and Lisa Cohen
RNA modulation, repair and remodeling by splice switching oligonucleotides
Acta Biochimica Polonica Vol. 51 No. 2/2004

Ribozyme-mediated RNA repair

Ribozyme-mediated revision of RNA and DNA -- Long et al. 112 (3): 312 -- Journal of Clinical Investigation: "The concept of ribozyme-mediated RNA repair was first demonstrated in Escherichia coli (2) and in cultured mammalian cells (7). "

DNA repair by AlkB

ScienceWeek: "AlkB enzymatically demethylates the DNA bases adenine and
cytosine, unlike the suicidal Ada and Ogt proteins, by oxidative
demethylation: the methyl group is converted to an hydroxymethyl
group which then leaves the base as formaldehyde."

Rad51p Rad54p Rad52p

Rad51p and Rad54p, but not Rad52p, elevate gene repair in Saccharomyces cerevisiae directed by modified single-stranded oligonucleotide vectors -- Liu et al. 30 (13): 2742 -- Nucleic Acids Research: "Synthetic single-stranded DNA vectors have been used to correct point and frameshift mutations in episomal or chromosomal targets in the yeast Saccharomyces cerevisiae. Certain parameters, such as the length of the vector and the genetic background of the organism, have a significant impact on the process of targeted gene repair, and point mutations are corrected at a higher frequency than frameshift mutations. Genetic analyses reveal that expression levels of the recombination/repair genes RAD51, RAD52 and RAD54 can affect the frequency of gene repair."

synthetic oligonucleotides

In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides -- Liu et al. 29 (20): 4238 -- Nucleic Acids Research: "Synthetic oligonucleotides have been used to direct base exchange and gene repair in a variety of organisms. Among the most promising vectors is chimeric oligonucleotide (CO), a double-stranded, RNA�DNA hybrid molecule folded into a double hairpin conformation: by using the cell�s DNA repair machinery, the CO directs nucleotide exchange as episomal and chromosomal DNA. Systematic dissection of the CO revealed that the region of contiguous DNA bases was the active component in the repair process, especially when the single-stranded ends were protected against nuclease attack."

DNA Size A Crucial Factor In Genetic Mutations, Study Finds

DNA Size A Crucial Factor In Genetic Mutations, Study Finds: "The researchers investigated it by offering bases of different sizes to the DNA polymerase I enzyme and measuring how accurately the enzyme made new DNA copies. About once every 10,000 to 100,000 times the enzyme will put in the wrong base, choosing for instance a G instead of a T to pair with an A. The rate that the enzyme accurately copies DNA is known as its efficiency.
These rare and random mistakes can cause genetic mutations. While we tend to heap negative connotations onto the term, some mutations create new traits that actually benefit the organism or yield no effect. These small-scale changes, collectively called genetic drift, play an important role in evolution, as does natural selection. "

Transvection

ScienceMatters @ Berkeley. Fly Guy: "In 1954, Nobel Laureate Ed Lewis, whom Levine calls the 'Einstein of flies,' proposed that a gene on one chromosome can directly affect the expression of its homologue gene on another chromosome, a process called transvection. However, the frequency of this 'crisscross' was unclear. Levine and Ronshaugen observed that, at least in the case of fruit flies, transvection is quite common.
'One possible explanation for transvection is maybe that it's used as a homeostasis mechanism,' Levine says. 'If an enhancer fails on one chromosome, the other chromosome can compensate. That way you make sure to get the right levels of expression.'"

Regulatory Genes & Enhancers

ScienceMatters @ Berkeley. Fly Guy: "Regulatory DNA, Levine explains, controls how and where a gene is expressed in a cell. Of the three types of regulatory DNA--enhancer, silencer, and insulator--'enhancers are king, activating gene expression in specific cell types for specific tissues,' he says. Scientists conservatively estimate that while the human genome has less than 30,000 genes, it may contain 100,000 enhancers at the minimum. So far, just 50 or so have been identified."

Gene Regulation in Eukaryotes

Gene Regulation in Eukaryotes: "Protein-coding genes have
exons whose sequence encodes the polypeptide;
introns that will be removed from the mRNA before it is translated [Discussion];
a transcription start site
a promoter
the basal or core promoter located within about 40 bp of the start site
an 'upstream' promoter, which may extend over as many as 200 bp farther upstream
enhancers
silencers"

genome.gov | Education Kit (Homepage)

genome.gov Education Kit (Homepage): "Human Genome Project released a free, multimedia educational kit for high school students and the interested public. . . the contents of the education kit were reformatted and are available on this website in two formats; download modules or online viewing."

hapMap

International Consortium Completes Map Of Human Genetic Variation: "In a paper in the Oct. 27 issue of the journal Nature, more than 200 researchers from Canada, China, Japan, Nigeria, the United Kingdom and the United States describe the initial results from their public-private effort to chart the patterns of genetic variation that are common in the world's population. The results provide overwhelming evidence that variation in the human genome is organized into local neighborhoods, called haplotypes, that usually are inherited as intact blocks of information.
At the project's outset in October 2002, the consortium set an ambitious goal of creating a human haplotype map, or HapMap, within three years. The Nature paper marks the attainment of that goal with its detailed description of the Phase I HapMap, consisting of more than 1 million markers of genetic variation, called single nucleotide polymorphisms (SNPs). The consortium is also nearing completion of the Phase II HapMap that will contain nearly three times more markers than the initial version and will enable researchers to focus their gene searches even more precisely on specific regions of the genome."

Epigenetics Alters How Our Genes Behave

'Epigenetics' Means What We Eat, How We Live And Love, Alters How Our Genes Behave: "The fact that gene behavior is far more malleable than once believed has critically shifted the scientific community's course in mining the human genome, said Jirtle. No longer are mutant genes sought as the sole cause of disease. "

Scientists Unpick Genetics Of First 15 Minutes Of Life

Scientists Unpick Genetics Of First 15 Minutes Of Life: "THIRA, ‘chaperones’ the early processes that take place once a sperm enters an egg, giving it a crucial role in the most fundamental process in sexually reproducing animals.

The absence or mutation of this gene in the maternal (mother’s) genome explains why eggs fail to produce a zygote – or early embryo - despite the presence of ‘healthy’ sperm.

The researchers looked at the genetic makeup of the sésame mutant and identified what is known as a point mutation in the HIRA gene -- showing that HIRA is the gene responsible for chaperoning the assembly of the sperm pro-nucleus."

Tsix transcription- versus RNA-based mechanisms in Xist repression and epigenetic choice.

Entrez PubMed: "Recent inquiries have revealed a surprisingly large number (>2500) of naturally occurring antisense transcripts, but their function remains largely undiscovered. A better understanding of antisense mechanisms is clearly needed because of their potentially diverse roles in gene regulation and disease. A well-documented case occurs in X inactivation, the mechanism by which X-linked gene expression is equalized between XX females and XY males. The antisense gene Tsix determines X chromosome choice and represses the noncoding silencer, Xist. In principle, Tsix action may involve RNA, the act of transcription, or local chromatin. Here, we create novel Tsix alleles to distinguish transcription- versus RNA-based mechanisms. When Tsix transcription is terminated before Xist (TsixTRAP), Tsix cannot block Xist upregulation, suggesting the importance of overlapping antisense transcription. To separate the act of transcription from RNA, we knocked in Tsix cDNA in the reverse orientation (Tsix(cDNA)) to restore RNA levels in cis without concurrent transcription across Xist. However, Tsix(cDNA) cannot complement TsixTRAP. Surprisingly, both mutations disrupt choice, indicating that this epigenetic step requires transcription. We conclude that the processed antisense RNA does not act alone and that Tsix function specifically requires antiparallel transcription through Xist. A mechanism of transcription-based feedback regulation is proposed."

Shibata S, Lee JT.
Tsix transcription- versus RNA-based mechanisms in Xist repression and epigenetic choice.
Curr Biol. 2004 Oct 5;14(19):1747-54.

Fitting In: Newly Evolved Genes Adopt A Variety Of Strategies To Remain In The Gene Pool

Fitting In: Newly Evolved Genes Adopt A Variety Of Strategies To Remain In The Gene Pool: "To determine the basis for the persistence of functional gene duplicates in the genome, three scientists at the Institute of Molecular Systems Biology at the Swiss Federal Institute of Technology in Zürich have collaborated on the largest systematic analysis of duplicated gene function to date. Using an integrative combination of computational and experimental approaches, they classified duplicate pairs of genes involved in yeast metabolism into four functional categories: (1) back-up, where a duplicate gene copy has acquired the ability to compensate in the absence of the other copy, (2) subfunctionalization, where a duplicate copy has evolved a completely new, non-overlapping function, (3) regulation, where the differential regulation of duplicates fine-tunes pathway usage, and (4) gene dosage, where the increased expression provided by the duplicate gene copy augments production of the corresponding protein.
Their results, which appear in the October issue of the journal Genome Research, indicate that no single role prevails but that all four of the mechanisms play a substantial role in maintaining duplicate genes in the genome."

Cold Spring Harbor Laboratory