The Darnell paper, Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps, published in the most recent issue of nature seems to be widely read by most people who care about RNA and or bioinformatics. This paper is important because Darnell, via high throughput sequencing of RNAs isolated by crosslinking immunoprecipitation, is able to differentiate between targets that are found by scanning the genome for the 6-8nt sequence complementary to the conserved "seed" sequence of an miRNA from those that show functional protein-RNA interaction.
Most bioinformatic approaches used to predict miRNA targets restrict their search to conserved target sites in the 3'UTR, the HITS-CLIP method, on the other hand, was able to find target sites that mapped to coding sequences, interenic regions, and non-coding RNAs. After identifying targets of miRNA, Darnell and his group wanted to see if the Ag0-mRNA/ Ago-miRNA interactions resulted in downregulation of the target mRNA. In order to determine this, HeLa cells were transfected with brain-specific miRNA (mir-124) and then used HITS-CLIP to determinte ago-mRNA clusters. According to the results, mRNA's that were bound by mir-124 were noticeably down regulated once transfected at the protein and mRNA level.
...Beyond the paper, i think it is interesting although not surprising that the HITS-CLIP method found targets in non-coding RNAs. In my opinion (which at this point probably isn't worth much given i have very limited experience with miRNA) might mean that these regulatory bits of RNA (miRNA) regulate other regulatory elements which in turn regulate the robustness of an even greater regulatory system. The Darnell paper restricted most (or all) of their analysis to conserved miRNA targets, however, I think that exploring the relationship between miRNAs and other non-coding RNA could offer insight into the feedback loops that govern how our genes are expressed.
Monday, July 27, 2009
Monday, July 20, 2009
Redefining MicroRNA Targets
The journal Current Biology published a report by Herve Seitz called Redefining MicroRNA targets. In this report, Seitz tries to reconcile the paradox between the thousands of puntative target sites regulated by a single microRNA and the fact that mutation of a single target mRNA of a given miRNA will rescue the mutant phenotype. Seitz finds it odd, or at least interesting that even though computational methods that elucidate a vast number of predicted target sites based on evolutionary conserved seed matches in 3' UTR regions in miRNA targets, that mutating just one of these predicted sites can result in a physiological change. Seitz believes that the discrepency between the large number of targets regulated by a miRNA and the ease with which a mutation in a single target mRNA can correct the defect can be explained by differentiating between real targets and pseudotargets. Seitz more stringent take on what really defines an miRNA target site looks beyond base-pair complementation.
An miRNA target site is usually defined based on the complementarity between the seed sequence of the miRNA and the mRNA it supposedly regulates. The seed sequence is very short and does not need to perfectly base pair with its target, and thus it makes sense that many mRNAs could be a potential target to any given miRNA. Now, on top of the fact that one miRNA can potentially regulate thousands of mRNA's, miRNA mediated regulation hardly causes changes in gene expression that exceeds 2 fold and this begs the question, how can RNAi or mutagenesis against only one miRNA target produce incredible phenotypic changes? Seitz proposes that a large proportion of miRNA binding sites act as competitive inhibitors for the miRNA and he refers to these as pseudotarget sites. The common denominator between bona fide target sites and pseudo target sits would be their ability to basepair with the same miRNA and this would be detected by most computational algorhithyms. The difference, however, between these two, according to Seitz, would be their sensitivity to small (2-fold) decreases in protein expression. Overall, Seitz claims that within the thousands of puntative or predicted miRNA targets based on imperfect basepairing in the 3'UTRs of mRNA, that only genes whose activity is sensitive to small reduction of protein levels can actually be labeled as a real bona fide miRNA target. Seitz uses let-7 as an example to explain why we see strong changes in phenotype when only one target is overexpressed instead of all those predicted. For example let-7 might regulate targets A,B,C, and D. If target A is knocked out or repressed via RNAi then the let-7 mutant phenotype is almost completely rescued even though targets B,C and D are not effected. Seitz postulates that some of these genes, therefore, must be pseudotargets for let-7. A pseudotarget, according to this report, is one that sequestors miRNA but is not sensitive to the small decrease in protein levels due to miRNA downregulation. Instead these targets tie up the miRNA in order to improve the repression of the small number of real targets. This postulation means that miRNA's and there mRNA targets regulate each other and that gene expression is based on the integration of expression information overall.
An miRNA target site is usually defined based on the complementarity between the seed sequence of the miRNA and the mRNA it supposedly regulates. The seed sequence is very short and does not need to perfectly base pair with its target, and thus it makes sense that many mRNAs could be a potential target to any given miRNA. Now, on top of the fact that one miRNA can potentially regulate thousands of mRNA's, miRNA mediated regulation hardly causes changes in gene expression that exceeds 2 fold and this begs the question, how can RNAi or mutagenesis against only one miRNA target produce incredible phenotypic changes? Seitz proposes that a large proportion of miRNA binding sites act as competitive inhibitors for the miRNA and he refers to these as pseudotarget sites. The common denominator between bona fide target sites and pseudo target sits would be their ability to basepair with the same miRNA and this would be detected by most computational algorhithyms. The difference, however, between these two, according to Seitz, would be their sensitivity to small (2-fold) decreases in protein expression. Overall, Seitz claims that within the thousands of puntative or predicted miRNA targets based on imperfect basepairing in the 3'UTRs of mRNA, that only genes whose activity is sensitive to small reduction of protein levels can actually be labeled as a real bona fide miRNA target. Seitz uses let-7 as an example to explain why we see strong changes in phenotype when only one target is overexpressed instead of all those predicted. For example let-7 might regulate targets A,B,C, and D. If target A is knocked out or repressed via RNAi then the let-7 mutant phenotype is almost completely rescued even though targets B,C and D are not effected. Seitz postulates that some of these genes, therefore, must be pseudotargets for let-7. A pseudotarget, according to this report, is one that sequestors miRNA but is not sensitive to the small decrease in protein levels due to miRNA downregulation. Instead these targets tie up the miRNA in order to improve the repression of the small number of real targets. This postulation means that miRNA's and there mRNA targets regulate each other and that gene expression is based on the integration of expression information overall.
Thursday, July 9, 2009
Tina Fey loves Ayn Rand
Tina Fey is a total objectivist and plugs Ayn Rand's philosophy in the wittiest ways! I love her and that her amazing show 30 rock opens up with a statue of Atlas!
Tina Fey loves Ayn Rand
The show 30 Rock is amazing and Tina Fey is a genius. In my opinion Tina fey seems to be an objectivist fan.
Wednesday, July 8, 2009
miRNA Introduction
Even though I plan on being a prolific neuroscientist oneday, right now I am lucky enough to work in the Pasquinelli lab at UCSD. Dr. Amy Pasquinelli is a total bad ass who knows everything about micro RNA's (miRNA) and below is a link to her lab (they need to update the picture on the homepage because I am not in it! :(
http://biology.ucsd.edu/labs/pasquinelli/Research.html
http://biology.ucsd.edu/labs/pasquinelli/Research.html
The word art appears to be enigmatic and is often ligated to a phrase that calls for its definition. What is art? for example, is a question that begs people to search for its meaning. Most people look beyond the stratosphere of reality- that which is known, and can never give a true and simple answer to that question. It seems unacceptable to view art as being true or false as “beauty is in the eye of the beholder”, however, I believe it is possible to find an objective answer if one accepts science as art.
The word science, like art is also enigmatic in its own right. The diametric connotations evoked by this word are like the chiaroscuro of a Da vinci painting as its contrasting elements create its meaning. In the light, or to most men under the sun who have never been in our labs, see our world as hidden behind an impenetrable wall of big words, dense text books, mathematical equations and sterile white lab coats. But to people who know what it’s like to have an insatiable appetite for knowledge, the word science scintillates with spectral fluorescence. Scientists to other scientists who love what they do are artists. A scientist, passionate about learning how the world in which he exists works, appreciates natural occurrences in life to be as impressive as Michaelangelo’s David or Rodin’s “The Thinker”.
My belief is that there is a lot of speculation about man and his existence and science and art are two ways that people try to find and express answers. Asking “what is art” is like asking “what is man”- two metaphysical questions that have answers rooted in objectivity. Some people cannot accept that existence is delimited by the immutable parameters set by nature and so they turn to some form of mysticism so they can keep their head in the clouds. My question to those people is, why look up to the sky when you can keep your eyes focused here on earth and learn about the one thing you were given in this world- your life. To me, the most beautiful sunset or even a Botticelli does not compare to the beauty that exists in the way our bodies and minds work. There is a whole unseen museum of artwork that live in the darks holes of our knowledge and science will illuminate those spaces.
The word science, like art is also enigmatic in its own right. The diametric connotations evoked by this word are like the chiaroscuro of a Da vinci painting as its contrasting elements create its meaning. In the light, or to most men under the sun who have never been in our labs, see our world as hidden behind an impenetrable wall of big words, dense text books, mathematical equations and sterile white lab coats. But to people who know what it’s like to have an insatiable appetite for knowledge, the word science scintillates with spectral fluorescence. Scientists to other scientists who love what they do are artists. A scientist, passionate about learning how the world in which he exists works, appreciates natural occurrences in life to be as impressive as Michaelangelo’s David or Rodin’s “The Thinker”.
My belief is that there is a lot of speculation about man and his existence and science and art are two ways that people try to find and express answers. Asking “what is art” is like asking “what is man”- two metaphysical questions that have answers rooted in objectivity. Some people cannot accept that existence is delimited by the immutable parameters set by nature and so they turn to some form of mysticism so they can keep their head in the clouds. My question to those people is, why look up to the sky when you can keep your eyes focused here on earth and learn about the one thing you were given in this world- your life. To me, the most beautiful sunset or even a Botticelli does not compare to the beauty that exists in the way our bodies and minds work. There is a whole unseen museum of artwork that live in the darks holes of our knowledge and science will illuminate those spaces.
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