To Code or Not To Code

An exploration into the vast unknown of long non-coding RNA

[nextgengenomics-blog]

Contrary to popular to popular belief, vegetables won’t actually kill you but, they may save your life one day. We all know that vegetables are loaded with lots of vitamins and minerals that are beneficial for our body and some of them are surprisingly delicious. However, one of the most hated vegetables is outshining all the rest.

Broccoli has always been a part of a healthy diet but is switching gears from the dinner table to the lab bench. A compound contained within broccoli (and other cruciferous vegetables) called sulforaphane has recently been associated with reducing long non-coding RNA (lncRNA) expression in prostate cancer cells.

Until recently, not a lot has been known about how our diet is impacting the expression of lncRNAs in our genome. Many lncRNAs have been associated with cancer progression but never with the decision between a tub of ice cream or a head of broccoli.

Researchers at Oregon State University set out to prove this association between sulforphane and cancer-associated lncRNA expression.

Figure 1. Broccoli contains glucoraphanin which is the precursor to sulforaphane. Once the tissues of broccoli are exposed by chewing, an enzyme called myrosinase is released to help hydrolyze and transform glucoraphanin. The final product is our desired product of sulforaphane and raphanin which has antimicrobial properties. 

To prove their hypothesis, the researchers started out by growing a cell line that contained healthy prostate cells and another that contained prostate cells effected by prostate cancer. Next, each cell line was treated with a specific solution of sulforaphane or a control solution to test the effects sulforphane had on each cell line. The cell lines were then left alone for a period of 24 hours and were harvested for further examination. Once the cells were harvested, a series of bioinformatics programs were used to sequence the lncRNAs and view their expression levels in the cell tissues. To strengthen their hypothesis further, the researchers performed a knockdown on a gene that encoded one of the lncRNA that showed the strongest association to a variety of cancers, LINC01116. To knockdown the gene, researchers infected prostate cancer cells with a small portion of double stranded RNA called small interfering RNA or siRNA. A guide strand then lead the siRNA to the gene of interest located on the messenger RNA. The siRNA is complementary to the gene of interest and can bind to the gene and mark it to be degraded thereby eliminating the gene so it cannot produce its gene product. More statistical analyses were complete before the results were calculated.

The results of the experiment turned out to be quite positive. Of the 407 expressed lncRNA in the prostate cancer cells, 119 of the lncRNA were significantly altered with the treatment of sulforaphane. In addition, 75.8% of the lncRNA in the prostate cancer cells were produced at higher levels in cancer cells when compared to normal prostate cells. In contrast, 64.5% of the lncRNA decreased in expression level after the treatment with sulforaphane.

But, these results don’t explain which lncRNA were up-regulated in prostate cancer cells or which lncRNA were down-regulated in sulforaphane treated prostate cancer cells. The team then identified a set of 7 lncRNAs that were overexpressed in prostate cancer cells and decreased with the sulforphane treatment. Interestingly, one of the lncRNAs studied showed the opposite pattern and was down-regulated in the prostate cancer cells but was up-regulated with the sulforaphane treatment. This could mean that this particular lncRNA (MIR22HG) plays a role in the cell cycle checkpoints that make sure mutated  cancer cells do not replicate and are degraded.

The question then became whether the other 7 lncRNAs were involved in pathways that are associated with cancer, or, if they had another unrelated function. After searching other sequence databases, the researchers found that the expression of these other 7 lncRNAs correlated with the suppressed expression of key regulatory molecules. Glyceraldehyde-3-phosphate Dehydrogenase (participates in the breakdown of sugar to form energy); Microtubule-associated Protein 1 Light Chain 3 beta 2 (participates in cell destruction); Beclin 1 (participates in cell destruction); H2A Histone Family, Member Y (regulates chromatin structure); and Ubiquitin A-52 (marks targets for degradation) all were the suppressed molecules. Now I know that’s a lot of jargon to take in but basically these molecules all play key roles in pathways cancer effects the most. This means that these lncRNAs not only are effected in prostate cancer but also other forms of cancer.

Figure 2. Prostate cancer is the most commonly diagnosed cancer in Canadian men. Its financial burden and social impact of many families present a need to further research in the field. Interestingly enough, prostate tissues are shown to absorb the most sulforaphane when compared to other tissues in the body. This is why researchers are exploring its function in prostate cancer.

These are promising results for the researchers and the public as many of us have been or know someone who has been effected by cancer. Having more information on what molecules do play a role in cancer is never a bad thing. Or is it?

After reading this paper I still remain a bit skeptical of the results and conclusions that were being drawn from the data. Cancer is a very complex disease and involves a lot pathways that all kind of team up to create one big perfect storm. It is possible that the sulforphane could have been acting on another pathway that in turn reduces the expression of the associated lncRNAs by coincidence. Many other research articles have been published with sulforaphane interacting with other molecules to stop cancer cells from growing. Also, the researchers never really explained how the sulforaphane suppressed the expression of the lncRNAs and didn’t even acknowledge the fact that there wasn’t concrete evidence for the connection.

However, I do think it’s a step forward in the right direction and an interesting interaction that I would have never really thought of before. A lot of work still needs to be done in this area of study before we can start making any clear statements and taking broccoli supplements. Right?

Wrong. Unfortunately, as I type this very post Amazon has an abundance of health supplement vendors selling 100% pure broccoli sulforaphane and it’s prime eligible. No way! Who needs to eat broccoli when you can just take a few pills and never have to worry about prostate cancer? While that mindset is shared by the same people that self-diagnose with WebMD, it’s not that simple. For starters, sulforaphane is unstable at temperatures above -20°C and the sulforaphane being sold is an altered form. So, before you click that “add to cart” button, take a second and do the research. Or you could just finish your vegetables.

Nonetheless, whatever method you choose to get your daily dose of sulforaphane and other daily vitamins is up to you. We may find out in the future the real reason why sulforaphane is suppressing these lncRNA which could lead to new chemopreventive methods for all types of cancer. All the more reason to put down that chocolate bar and pick up some broccoli.

For further information, check out the links below:

Article

Article

[nextgengenomics-blog]

One of the hardest things that I’ve had to wrap my head around during my exploration of long non-coding RNA (lncRNA), is the fact that only 2% of our genome is produced into proteins while the other 98% does not produce proteins. There is so much we still don’t know about ourselves despite having come so far with only 2% of the knowledge. Recently we have made great discoveries within the 98% but much of its mysteries still remain unsolved. However, one project is looking to change this and has with their latest comprehensive atlas of human lncRNA.

FANTOM (Functional ANnoTation Of the Mammalian genome), is a worldwide collaboration project to identify all the functional elements of the mammalian genome. Over the past 17 years, FANTOM has come out with 5 projects all in relation to the function and identification of these elements in the genome. With their most recent project FANTOM5, collaborators set out to investigate how our genome encodes the diverse number of cells we have that make up our bodies. Within this project for the first time, a comprehensive atlas was created upon the discovery of nearly 28,000 lncRNA that were found in the human genome.

The atlas of the 28,000 lncRNA summarizes their expression over a wide range of tissues and cell types in humans. By using their atlas and other genomic data they generated, the researchers were able to conclude that of the 28,000 lncRNA, 19,175 of these lncRNA were functional. To put that into perspective, we almost have as many functional lncRNA as we do protein coding genes in our genome (» 20,000) and lncRNAs are only a small part of the 98% of the non-coding portion of the human genome.

Thanks to the atlas, this further strengthens the argument of lncRNA having a functional role in our genome and not just being non-functional “junk RNA”. It also provides us with a better resource to build upon our every growing knowledge of lncRNA.

For more information, check out the links below:

Article

[nextgengenomics-blog]

If you look closely, the portrait is made up of the nucleotide sequence of the gene that is responsible for producing the alanine transfer RNA that Robert characterized in yeast (Artwork by Andrew Spindler).

The man portrayed in the portrait above is not just some man with a great pair of glasses. He is a Nobel Prize winner, a biochemist, and the founder of one of the first non-coding RNA. Robert W. Holley was an American biochemist that sequenced and proposed the structure of the alanine transfer RNA in yeast. Transfer RNA (tRNA) are non-protein coding molecules of RNA that are responsible for the transport of an amino acid sequence to a ribosome which then produces a protein.

While Robert’s work did not focus on the field of long non-coding RNA, his work helped pave the way of determining various RNA molecule structures and uncovering the importance of these RNA molecules. Without the contributions by Robert and many other scientists alike, most long non-coding RNA would still be deemed molecular junk today.    

 Fun Fact: Holley used a total of 140kg of baker’s yeast to collect only a measly 1g of purified alanine tRNA to use for the sequence and structure determination.

More information on Robert can be found here     

[nextgengenomics-blog]

Rett syndrome is a rare X-chromosome linked disorder that effects over 350,000 women worldwide a year and is sometimes misdiagnosed as cerebral palsy or autism. Unlike autism and cerebral palsy however; Rett syndrome is caused by a mutation of the methyl-CpG-binding protein gene (MeCP2). MeCP2 encodes a methyl-CpG-binding protein which is known to be crucial in the development and function of neurons. Rett syndrome also differs from cerebral palsy and autism in the way it is inherited from generation to generation. The most common disorders are inherited in a homozygous recessive fashion meaning 2 mutated copies of the same gene are needed to produce the disorder. As a heterozygous dominant disorder, Rett syndrome only needs one mutated copy of the gene on the X-chromosome to cause the disorder instead of 2 mutated copies.

While Rett syndrome mostly occurs in women, men can also be effected by this disorder but in cases that are rarer. Unlike women, men have an X and a Y chromosome.  Because men only have one X-chromosome, when the MeCP2 gene is mutated, it does not have a backup X-chromosome to compensate for the mutation and results in death. In women, however; the functioning allele that is located on one of the X-chromosomes, can compensate for the mutated allele and results in Rett syndrome instead of death. What also makes Rett syndrome more common in women is that cells in women undergo a process called X-inactivation that plays a key role in the initiation of Rett syndrome.

X-chromosome inactivation is a process in which one of the copies of the X-chromosome that are present in female cells is inactivated (See figure 1). These cells initially start off each containing a pair of X-chromosomes that are passed down from the mother and father. During embryonic development either the paternal (father) or maternal (mother) X-chromosome will be inactivated. This ensures that men and women have equal X-chromosome gene products since males only have one X-chromosome copy. The decision of which X-chromosome will be inactivated is completely random and will vary from cell to cell. With different X-chromosomes being active in different cells, this means that the genes being expressed will vary as well. One of the more well-known examples of this process is seen every day and you might not have even known.

Figure 1. The X-inactivation process plays a major role in many X-chromosome linked disorders.

Many of us have cats at home and never realized that they’ve been hiding something from us all along. Lurking deep in the nucleus of your cat’s cells is the secret they’ve been hiding all along, an extra X-chromosome. That extra X-chromosome is never talked about but, is seen in the pattern of colour of your cat’s fur. While all female cats undergo this process, it is most commonly seen in the colouring pattern of Calico cats. Calico cats are known for their tortoiseshell colouring pattern made up of orange and black fur. The gene that encodes the colour of the cat’s fur is located on the X-chromosome and comes in 2 versions on the gene, one that codes for black fur and another that codes for orange fur. When one of these X-chromosomes is inactivated, the other colour will be expressed. Since the X-chromosome inactivation is random and varies from cell to cell, we get these characteristic patches of orange and black that is seen on the cat (See figure 2). While this process has been well documented to occur in female mammals, the actual mechanism of how the X-chromosome is inactivated is starting to come into focus.

Figure 2. In male Calico cats, the colouring pattern is straight forward because they only have one X-chromosome. In heterozygous female cats (far right), cells can either display orange or black depending on which X-chromosome is inactivated in the cell. This gives us the patched fur pattern. 

With the help of next-generation sequencing for uncovering the blueprint to our genome, a stretch of long non-coding RNA(lncRNA) was found to play a role in the X-inactivation mechanism. The XIST gene is located on the X-chromosome and is responsible for encoding an RNA transcript which is a stretch of RNA sequence that has regulatory functions. Prior to the inactivation of one of the X-chromosomes, XIST is expressed at very low levels in both copies. During the inactivation process, however; one copy of the X-chromosome is inactivated with the up-regulation of the XIST gene and XIST transcript. The XIST transcript covers the entirety of the X-chromosome and blocks other molecules from binding and interacting with the X-chromosome (Figure 2). The XIST transcript then starts to change the structure of the inactive X-chromosome and forms a ball of heterochromatin called a Barr body. This Barr body is then conserved and remains in that form throughout the lifespan of the cell. It is not completely known how the XIST transcript covers and changes the structure of the X-chromosome but it is known that the X-chromosome becomes inactivated by this process. The other XIST gene on the active X-chromosome is down regulated by another non-coding RNA called TSIX. TSIX is antisense to XIST meaning that it’s sequence is the same as XIST but in reverse (just like its name). Since TSIX is antisense to XIST, it is complimentary and can bind to the XIST gene. Binding to the XIST gene blocks other molecules from interacting with the XIST gene and silences the gene making the X-chromosome active. Other molecules and transcripts are associated with this process but are not as well studied as TSIX.

Knowing that the XIST gene and transcript play a major role in X-chromosome silencing then opens up an opportunity for a possible exploit in X-chromosome linked diseases. In the case of Rett syndrome, women with the disorder do have a defective copy of the MeCP2 gene on the active X-chromosome but, they also have an fully functional copy of MeCP2 on the inactive X-chromosome. What if there was a way to reactivate the silenced X-chromosome which would allow the functional copy of MeCP2 to compensate for the mutated MeCP2?

Researchers at the Fred Hutchinson Cancer Research Center in Seattle set out to do just that as they looked to ways reactivate MeCP2 in individuals suffering from Rett syndrome. By down regulating the expression of XIST in the inactivated X-chromosome, the X-chromosome would become active again. The researchers pinpointed target genes on the inactive X-chromosome that would work together to suppress XIST gene expression.

In total, 30 genes were found from previous research that play a role in the awakening of the silenced X-chromosome and function of MeCP2 gene. The screens consisted of developing cell lines that had a specific tag associated with the MeCP2 gene so that researchers were able to view if the MeCP2 gene was on the active or inactive X-chromosome. This specific tag included an antibiotic resistance to hygromycin and a luciferase gene which encodes bioluminescence. Next they injected embryonic mice cells with the tagged MeCP2 gene and grew them in environments that selected for hygromycin resistance to make sure they had cells that accepted the tagged gene. They now screened these cells and if the fluorescence was seen in the inactive X-chromosome, they would treat them with a hypomethylating agent (See figure 3).

Figure 3. Of those 30 genes, a subset of only 4 of those genes were found necessary to reactivate the MeCP2 gene on the inactive X-chromosome. The hypomethylating agent that was used is called 5-azacytidine (5’-AZA). 5’-AZA would add methyl groups to genes involved with XIST expression to block other molecules from interacting with them which would inactivate XIST expression. Inactivating XIST would then lead to reactivation of the X-chromosome and MeCP2 gene.

The researchers then decided to apply this method of treatment in mice models. They were able to reactivate MePC2 in mice with Rett syndrome and help alleviate symptoms of the disorder. However, one unfortunate setback of this method is that they also reactivated all the genes on the X-chromosome which isn’t ideal. Reactivating all the genes resulted in death in mice models but with partial reactivation of the MeCP2 gene. This could be due to other mutated genes on the formerly inactive X-chromosome that led to the death of the mouse. Further mice trials are needed to improve the specificity of the compounds being used before heading in to pre-clinical trials with this method of treatment.

Long non-coding RNAs are vital components in many processes of humans from XCI to serious disorders such as Rett syndrome. Understanding how these lncRNA interplay with other molecules in our body is key to our discovery of future therapeutic targets. With this technique, we could apply it to many other diseases by reactivating genes at specific locations on the X-chromosome such as muscular dystrophy which is another X-linked genetic disorder. This technique could also be applied to alleviate symptoms for adults already suffering from various X-linked disorders. So, stop sleeping on the X-chromosome because it when it wakes up, it could surprise you with what it can do.

For further information check out these articles:

Reactivating in Mice Models

MeCP2 in Rett syndrome

Article

[nextgengenomics-blog]

Affecting nearly 1 in every 6000 births, spinal muscular atrophy (SMA) is one of the most common genetic muscle-wasting disorders among people around the world. SMA is an autosomal recessive disorder meaning two copies of the mutated gene are needed for any symptoms to occur. These symptoms occur from the survival motor neuron 1 gene (SMN1) being mutated and effecting the production levels of survival motor neuron proteins (SMNP). SMNPs are pivotal for the development of motor neurons in our body and when there are lower levels being produced at the improper time, muscles under-develop. Researchers at Johns Hopkins University have found a possible solution to help relieve symptoms of SMA by targeting a long non-coding RNA (lncRNA).

 As the researchers were trying to discover a reason why different levels of SMNPs were being produced, they came across a survival motor neuron gene (SMN) that encoded a lncRNA that was expressed during early muscle development. During normal early development, the SMN lncRNA was expressed at low levels to allow for regular production of SMNPs. In later development, the SMN lncRNA is expressed more which reduces the production of SMNPs. With SMA, the pattern is reversed which leads to muscle-wasting or even death. In theory then, if SMN lncRNA was somehow regulated and the expression pattern reversed, some of the symptoms of SMA would be relieved. In fact, that’s exactly what the researchers set out to do with a tag team of compounds including one of the newest drugs on the market.

The first drug Spinraza (Nusinersen), is a newly FDA-approved drug that is currently the only drug on the market for SMA. The drug is an antisense oligonucleotide which is a strand of DNA or RNA that is complementary to another known sequence. What is special about Spinraza is that it induces splicing where it binds to the known sequence. Spinraza binds to the highly similar SMN1 gene called SMN2 and cuts it at a specific location. The cut that is made converts the SMN2 gene into a SMN1 gene that if fully functional. The second compound used was also an antisense oligonucleotide-A (ASO-A) that interacts with the SMN lncRNA. ASO-A binds to the lncRNA and reduces its expression which up-regulates SMNP production.

Having Spinraza create a functional SMN1 gene and ASO-A block SMN lncRNA from down-regulating SMNP production is a great one-two punch. To prove this, mice models were tested upon and mice who were treated with both compounds lived longer and gained muscle mass when compared to mice who were treated solely with Spinraza or other compounds.

Even though this dynamic duo of drugs does not cure SMA all together, it is a start to understanding the disorder and what role lncRNA plays. Hopefully, further research and development can be done to not only to better the drugs but also to reduce the cost of Spinraza which currently stands at a hefty price tag of $750,000 per year.

For further information, feel free to check out the article below:

Article

New York Times article on the cost of Spinraza:

Article

[nextgengenomics-blog]

Step aside ribosomes, there’s a new RNA molecular machine in town! Well, not actually, but one that is built very similar and is just as interesting. Researchers at the Los Alamos National Laboratory in New Mexico have taken an incredible step forward in the world of long non-coding RNAs with their discovery of the structure of COOLAIR.

COOLAIR is a long non-coding RNA (lncRNA) that is responsible for the timing of flowering in plants during the spring time. It can sense how long the plant has been exposed to the cold temperatures and turn itself off to keep the flower from blooming at the wrong time. Acting in conjunction with a protein called FLC (Flowering Locus C), COOLAIR is the head of the control center when it comes time for the plant to bloom.

So, what exactly is so cool (no pun intended) about this lengthy stretch of RNA? In their study, researchers were able to create a 3D structure of COOLAIR and discover that the structure resembled that of a ribosome which is unheard of for a stretch of RNA. Very few lncRNA have been constructed in 3D let alone one that has this staggering resemblance. Having this structure can help us understand other lncRNAs with similar functions in other organisms, especially in regards to epigenetics. Epigenetics plays a huge role in embryo development and the diseases that can occur during that stage of life in humans.

I highly suggest checking out the article for a bit more background on the discovery if it piques your interest.

Enjoy!

-Andrew

Links:

Article

Youtube 

[nextgengenomics-blog]

For Your Information

Hi there, my name is Andrew and if you’ve found this blog you either share a passion for genes like I do or, Google has led you very astray. Nonetheless, if you share an interest in the world of genetics and how it’ll impact your life, you’ve come to the right place.

Before we get into the reason why I created this blog, I feel like I should introduce myself a little bit more. I was born in the heart of Saint John, New Brunswick and moved to the pizza and used car dealership capital, Lower Sackville, Nova Scotia at the age of 4. I’ve always had an interest in doing something in the scientific field from an astronaut to a hockey scientist (I was like 6 years old okay…). The end goal was always in the pursuit of understanding how the world around us works. After doing some growing up, I narrowed it down to being a scientist of some sort and I packed my things and came to the lovely town of Freddy Beach to pursue just that. As a fourth-year biochemistry student now, I’ve been exposed to a wide variety of topics and developed a love for genetics and molecular biology.

While most of my weekdays are filled with homework and assignments, the weekend is when I’m able to entertain some of my other interests. I enjoy playing hockey, annoying my roommates with my extensive music taste, watching lots of movies, and traveling whenever I get the chance.

Now that that’s out of the way, let’s get into the reason why you’re really here, Science! In particular, my blog will be talking about the vast unknown that is long non-coding RNAs. Usually what comes to mind first when we hear non-coding RNA is a RNA that is not transcribed into a protein. If this is true, then why on earth do we still code for these RNA? The answer to that question is one which I’ll be exploring in my blog.

In the past decade or so, the focus of most research has been on the 2% of the genome that is the protein coding region and has ignored the other 98% of the genome that is non-coding. Thankfully, in recent years, scientists have started to pay more attention to this non-coding region and discover the purpose of this region. This new research has suggested that these regions producing long non-coding RNA have multiple functions that are vital to cells in our body and play roles in a lot of diseases such as certain types of cancer. Over the course of this semester I will be diving into the literature to explore these functions and how they impact not just the scientific community but also you, the reader.

Hopefully something I’ve mentioned so far has gotten you as curious as I am to explore this rapidly growing field of study. If so, come along and join me as we explore the vast unknown of long non-coding RNA.

My dad and me between the Silfra fissure at the Thingvellir National Park in Iceland.