The Subliminal Messenger - a blog about MicroRNA and the Mind

UCSD Neuroscience

2010-03-08T12:22:00.000-08:00

I am so so so happy to say that I just recently got accepted into UCSD's Neuroscience Graduate Program!!!! So over the next 5-6 years I anticipate that this blog will become very active and informative since I will be reading a countless number of papers. I am really excited to set up my lab rotations and right now I am looking at the Ed Callaway Lab at the Salk institute as well as the Pam Reinagel lab and Anirvan Ghosh lab at UCSD!!!

To my 3 followers :)

2010-01-12T12:57:00.000-08:00

Hello to the few of you who actually read my blog! I have not updated for a while because I have been applying to grad schools and enjoying some leisure time! Now that I have received invitations to interview at different Neuro Ph.D. programs I feel inspired and excited to start reading again even though I am not in a lab at the moment. Recently I came across a paper published in Science called Reproducibility Distinguishes Conscious from Nonsconscious Neural Representations and I am looking forward to discussing the findings from that paper as well as others over the next few weeks!

MicroRNAs and disease

2009-11-01T13:00:00.000-08:00

A new database has recently emerged that links hundreds of human diseases to the aberrant expression of microRNAs.
http://www.miR2Disease.org/

Active turnover modulates mature microRNA activity in C.elegans

2009-10-28T10:04:00.001-07:00

A paper published in a september issue of nature implicates 5-3' exoribonuclease XRN-2 as a regulator of functional mature microRNAs in c. elegans. The biogenesis pathway of microRNAs has been characterized up until the point where the mature strand is loaded into RNA binding protein Argonaute forming the RNA Induced Silencing Complex. This complex acts to either signal target mRNA for degradation or translational repression. Understanding the microRNA biogenesis pathway is incredibley important for many reasons, and elucidating the fate of mature microRNAs after they have been processed is probably most important as the mature form of some microRNAs like let-7 are shown to be downregulated in various cancer cells.

In this publication, the authors show that XRN-2 depletion leads to an increase in mature let-7 in vivo and in vitro. For there in vivo assay, larval worms were synchronized and fed bacteria that expressed double stranded RNA in order to knockdown different nucleases for there screen. Worms were let-7 n2853 mutants that rupture through their vulva due to less let-7 being made relative to wild-type. This particular let-7 allele was used for the RNAi screen because any nuclease that suppressed the bursting vulva phenotype would reveal a negative regulator of let-7. From the screen the authors found that 98% of the worms showed suppression on XRN-2. Northern blot analysis confirmed that there was an increase in mature levels of let-7 on XRN-2 RNAi without any affects on the pri and pre forms of microRNAs. Following there in vivo assay, the authors developed a biochemical approve to recapitulate what they showed from there screen. Using worm lysate that was both positive and negative for XRN-2, the researchers revealed that microRNA degradation was dependent on XRN-2 being present in the lysate. Their expirements evinced a role for XRN-2 specific for degradation of single stranded mature microRNAs that are not bound to any targets. Overall this finding is very significant if in fact XRN-2 does cause downregulation of active microRNAs. However, other publications done in other models show XRN-2 to have completely different roles. In arabidopsis, XRN-2 degrades the loop sequence of pre-miRNA without affecting levels of the mature form. In yeast, XRN-2 has been showed to be in involved in clearing away inactive tRNAs and finally, in HeLa cells, XRN-2 was shown to be a nuclear protein that degrades miRNA transcripts. It will be interesting to see other researchers can confirm that XRN-2 is affecting mature microRNAs as this could possible lead to new theraputic measures for diseases caused by aberrant microRNA expression.

You and Your Research

2009-09-06T12:18:00.000-07:00

Advice to the young scientist...
http://www.cs.virginia.edu/~robins/YouAndYourResearch.html

Mobile Elements, "Jumping Genes", and non-coding RNAs Responsible for Genetic Diversity in the Brain

2009-08-23T14:02:00.000-07:00

The question "what is man?" is very general, and generally speaking, very hard to define. Conflicting answers to this question among people are likely to emerge, as answers given to any question are often experience dependent and manifest themselves within the context of a given individuals system of values.Defining the nature of human beings as it pertains to existence on both a metaphysical and epistemological level is challenging. From an ontological angle, subjectivity is an inexorably contaminent of perception that makes the clear elucidation of the essence of human existence impossible thus far. Epistimologically, characterizing man is challenging because the paucity of our knowledge concerning human nature creates an obstacle for scientists and philosophers who have consecrated a life in approaching this question in novel ways.
One such example of a scientist interested in the cognitive attributes unique to man at such a high degree of complexity, is research scientist Fred Gage at La Jolla's Salk institute. Gage has been working on trying to pinpoint the molecular events that cause individuals to have a large spectrum of neural diversity based on the difference in protein expression profile's across neuronal cell subtypes. Understanding the differences in protein expression and they ways that these differences arise, Gage believes, will offer insight into the relationship between the generation of neuronal diversity and complex cognitive functions like conscioussness and synaptic plasticity. In Gage's work, to be published in Nature, he and his team of scientists have found reasons to believe that mobile genetic elements coupled with different mechanisms that post-transcriptionally control how genes are regulated are responsible for the rich variety of neuronal cell types found in the human brain that is unlike any other organism. The mobile elements that Gage holds responsible for this diversity are LINE (L1) Retrotranspositions or long interspersed nuclear elements.
LINE retrotransposons are mobile pieces of DNA that generate random and reversible insertions through a copy and paste mechanism. The L1 gene has an internal promoter in its 5' UTR to drive expression of its two gene products from its two open reading frames. ORF 1 encodes and RNA chaperone protein and ORF 2 encodes the dual action endonuclease/reverse transcriptase enzyme. By making a nick in DNA via the endonuclease the L1 element is able to make an insertional copy of itself via the RT enzyme anywhere in the genome with the consensus sequence 5'TTTA3'. These L1 insertions are non bias, thus, they can be used to generate a large number of mutations within any single cell population. The mutations caused by L1 mediated insertions and how they might lead to brain specific phenotypes affecting cognition seems to be only speculative rather than conclusive at the moment. However, L1 retrotransposition mediated mutagenesis combined with several different systems of gene regulation can offer insight into how neurons are so diverse even within a single subpopulation of differentiated cells. One example of a post-transcriptional gene regulation system that might be working in conjunction with L1 elements to produce the incredibly high level of complexity seen in human brain cells are small non-coding regulatory RNAs called (of course) microRNAs. MicroRNAs reveal ways in which post-transcriptional gene expression can be modified and controlled in an epigenetic fashion or without changing any underlying DNA sequence. A possible example that conveys how mobile genetic elements, neuronal diversity and microRNA regulation are related is the gene SOX-2. According to Gage, L1 retrotransposable elements are silenced in neural stem cells due to SOX-2 mediated transcriptional repression. Downregulation of SOX-2 can lead to epigenetic changes that trigger neuronal differentiation. Besides Gage's observation about SOX-2, scientists in the Pasquinelli lab have found SOX-2 to be a direct target or suppressor of micro RNA let-7 in C. elegans. According to scientific literature, let-7 expression is strongly downregulated during neural differentiation of EC cells. If SOX-2 is really a direct target of let-7 then this might mean there is a regulatory feedback loop at work governing the timed expression of let-7 and differing modifications responsible for nueronal diversity such that differentiation and genetic diversification is coupled. For example, if let-7 negatively regulates SOX-2 and SOX-2 silences L1 retrotransposistion via transcriptional repression then there might be communication between mobile genetic elements and let-7 mediated by SOX-2. This hypothetical mechanism would reveal the sort of control at work when a neuron differentiates with regaurd to when genetic modifications are occuring during development of the cell.

Bona-fide miRNA Targets

2009-07-27T18:57:00.000-07:00

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.

Redefining MicroRNA Targets

2009-07-20T10:57:00.000-07:00

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.

Tina Fey loves Ayn Rand

2009-07-09T15:42:00.000-07:00

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

2009-07-09T15:25:00.000-07:00

The show 30 Rock is amazing and Tina Fey is a genius. In my opinion Tina fey seems to be an objectivist fan.

miRNA Introduction

2009-07-08T17:54:00.000-07:00

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

2009-07-08T14:45:00.000-07:00

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.