Widespread RNA and DNA Sequence Differences in the Human Transcriptome

Inamul Hasan.Madar Gaseous Ion Laboratory, Korea University, Korea

http://www.nature.com/news/2011/110525/full/473432a/box/1.html

1000 Genome Project

First project to sequence the genomes of a large number of people based on human genetic variation. GOAL The 1000 Genomes Project is to provide a resource of almost all variants, including SNPs ,structural variants, and their haplotype contexts. This resource focus on almost all variants that exist in regions found to be associated with disease. The genomes of over 1000 unidentified individuals from around the world will be sequenced using next generation sequencing technologies. The results of the study was publicly accessible to researchers worldwide.

Strategy
1)Whole-genome sequencing of 180 samples, 2) Whole-genome sequencing of 2 mother-father-adult child trios, 3)1000 gene regions in 900 samples

http://www.1000genomes.org/about

International HapMap Project

Catalog of common genetic variants that occur in human beings. What these variants are, where they occur in our DNA, and how they are distributed among people within populations and among populations in different parts of the world. Sequences will differ once in every 1,200 bases (Figure ). One person might have an A at that location, while another person has a G, or a person might have extra bases at a given location or a missing segment of DNA.

B cell

B cells are lymphocytes that play a large role in the humoral immune response . Functions of B cells are to make antibodies against antigens, perform the role of antigen-presenting cells (APCs) and eventually develop into memory B cells after activation by antigen interaction. B cells are an essential component of the adaptive immune system. "B", in B cell, comes from the bursa of Fabricius in birds, where they mature.

ADAR- Adenosine Deaminase, RNA-specific

Double-stranded RNA-specific adenosine deaminase is an enzyme that in humans is encoded by the ADAR gene. RNA editing by site-specific deamination of adenosines. This enzyme destabilizes double stranded RNA through conversion of adenosine to inosine. Mutations in this gene have been associated with dyschromatosis symmetrica hereditaria. Alternate transcriptional splice variants, encoding different isoforms, have been characterized A-to-I editing, arising from the fact that I behaves as if it is G both in translation and when forming secondary structures. These effects include alteration of coding capacity, altered miRNA or siRNA target populations, heterochromatin formation, nuclear sequestration, cytoplasmic sequestration, endonucleolytic cleavage by Tudor-SN, inhibition of miRNA and siRNA processing , and altered splicing.

APOBEC-1-C->U-editing enzyme
APOBEC1 apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1.Which is cytidine deaminase enzyme This holoenzyme is involved in the editing of C-to-U nucleotide bases in apolipoprotein B and neurofibromatosis-1 mRNAs

http://www.ncbi.nlm.nih.gov/gene/339

RNA Editing
RNA editing is a post-transcriptional modification of RNA and markedly increases the complexity of the transcriptome. RNA editing occurs in the nucleus, as well as in mitochondria and plastids. To date such changes have been observed in prokaryotes, plants, animals and virus. The diversity of this widespread phenomenon includes nucleoside modifications, nucleotide additions and insertions, either in coding or non-coding sequences of RNA, which can occur concomitantly with transcription and splicing processes. Most of the RNA-editing processes, however, appear to be evolutionarily recent acquisitions that arose independently. The diversity of RNA editing mechanisms includes nucleoside modifications such as cytidine (C) to uridine (U) and adenosine (A) to inosine (I) deaminations, as well as nontemplated nucleotide additions and insertions. RNA editing in mRNAs effectively alters the amino acid sequence of the encoded protein so that it differs from that predicted by the genomic DNA sequence[1].

http://bioinfo.au.tsinghua.edu.cn/dbRES/index.php

RNA Editing Type

+---C to U RNA editing +---A to G RNA editing +---U to C RNA editing +---G insertion RNA editing +---C insertion RNA editing +---G deletion RNA editing +---A to C RNA editing +---G to C RNA editing +---G to A RNA editing +---U insertion RNA editing +---A insertion RNA editing +---C/U insertion RNA editing +---AA insertion RNA editing +---A to U RNA editing +---GG to AA RNA editing +---AA to GG RNA editing

http://bioinfo.au.tsinghua.edu.cn/dbRES/browse_edittype.php?edtype=NULL&geneName=NULL

http://en.wikipedia.org/wiki/RNA_editing

Editing by deamination
C-to-U editing is with the apolipoprotein B gene in humans. Apo B100 express in liver & apo B48 in intestines. B100 has a CAA seq edited to UAA, a stop codon, in the intestines. It is unedited in the liver.

A funny thing happened on the way to the ribosome. That's the essence of a controversial paper concluding that messenger RNA the molecular middleman that carries information from a cell's DNA to its protein-making machinery is routinely and systematically altered by unknown mechanisms before its genetic instructions can be read. The paper, published in Sciencelast week (M. Li et al. Science doi:10.1126/science.1207018 ; 2011), is already drawing pointed reviews from computational biologists, who cite possible flaws that could undermine the authors' claims. If verified, the findings would require a rewrite of the 'central dogma' of molecular biology, which posits that the RNA transcripts that carry genetic information to the ribosome, where they are used as templates for protein assembly, are generally faithful matches to the original DNA. A revised version of the picture would include an 'RNA editing' step along the way, which replaces individual letters in the genetic code and changes the resulting proteins . Such a step would allow cells to generate much more diversity from the standard DNA tool kit than previously thought. Vivian Cheung of the University of Pennsylvania in Philadelphia led the work, which involved examining the RNA transcripts and DNA sequences of 27 people who were sequenced in the 1000 Genomes Project and the International HapMap Project. The team found more than 10,000 sites in exons regions of messenger RNA that have been transcribed from DNA in which the DNA and RNA sequences did not match. The same mismatches occurred in different people, suggesting that they were not random mistakes in transcription. Cheung's team also found proteins made from the 'mismatched' RNAs. "Once we saw that these differences were translated into protein sequences, we were pretty certain that they were biologically derived," Cheung says. RNA editing a process that changes the identity of an RNA base after it has been transcribed from a DNA sequence is not a new discovery. An enzyme called ADAR, for instance, induces mismatches in human cells by replacing the base adenosine with another molecule that is then read as guanine when the RNA is used to code for a protein. RNA editing also occurs in plants and human parasites. But the extent of RNA editing posited by the Science paper is extraordinary; its authors estimate that each person has about 1,065 mismatches sites the authors call "RNA DNA differences", or RDDs. Some of the mismatches involve base changes that are not produced by known RNA-editing mechanisms, suggesting that undiscovered mechanisms are at work.

http://www.gene-watch.org/blog/post/Evidence-of-altered-RNA-stirs-debate.aspx

"This suggests a completely different layer of gene regulation at the RNA level," says molecular biologist Kazuko Nishikura at the Wistar Institute in Philadelphia. "The big challenge now is to sort out the molecular mechanism for how these RNA sequence alterations can be achieved." Others remain skeptical. Comparative genomicist Lior Pachter at the University of California, Berkeley, has studied how the high-throughput sequencing machines that Cheung's team used to sequence RNA make systematic errors when sequencing DNA and RNA. He says that some of Cheung's mismatches occur at sites that are prone to systematic RNA sequencing errors, but others do not. And in a post on the blog 'genomes unzipped' on 20 May, Joe Pickrell, a graduate student working with human geneticist Jonathan Pritchard at the University of Chicago, Illinois, described another potential source of error. Pickrell said that multiple regions of similar DNA in the human genome can make it difficult to trace the origin of a short stretch of RNA to a specific DNA sequence, creating the illusion of DNA RNA differences. "If the authors are accidentally attributing RNA from two different regions of the genome to the same DNA region, they could falsely infer RNA editing," Pickrell said. "I think many of their results could be the result of errors in identifying the correct genomic origin of their sequencing reads." Other researchers are combing through their own data and waiting to see the results of follow-up work that will determine whether the concerns raised by Pachter, Pickrell and others are valid. Meanwhile, Chueng says, "we are glad to see that our colleagues are already using our data". If confirmed, Cheung's work has important implications for biology and for the way that researchers study genomics. Chris Gunter, director of research affairs at the HudsonAlpha Institute for Biotechnology in Huntsville, Alabama, says that RNA editing might have implications for the genetic origins of disease, if it turns out that the control of how much editing occurs is inherited. "This could make our jobs as geneticists more problematic and more interesting," she says

DARNED RNA editing in humans. A-to-I editing, a few C-to-U instances are also included in the DARNED dataset