Hope you all have been having a wonderful summer so far (I sure have!), and I just wanted to share what I’ve been/ what I’m going to be up to this summer =)
To begin, I’m Prakriti (or Parki), a rising junior, and I have the amazing privilege of working at the Phillip Sharp Lab at the Koch Institute for Integrative Cancer Research at MIT. The Sharp Lab is a pure biology lab, aiming to understand the very foundations of molecular biology, with an emphasis on anything RNA-related and the processes of alternative splicing and RNA interference. Given the fundamental nature of the research, the research has significant downstream consequences in understanding various disease mechanisms and pathology and will be useful in coming up with innovative treatments to them. Thus, it is extremely exciting for me to be surrounded my grad students and postdocs who are going after such interesting questions, constantly discussing their research, and to even be a part of the research process!
I’ll talk about what I’m doing and some other lovey-dovey things about lab <333 (maybe I’ll save that part for a second blog)
What I’m slaving at pipetting and tissue culture for <333- Overview
1) Understanding how RbFox2 is co-regulating alternative splicing that leads to Nonsense-Mediated Decay for RNA binding proteins.
2) Understanding how chromatin-remodeling influences alternative splicing patterns regulated by RbFox2.
3) Cloning Setd2 and Dot1L (histone methyltransferases), and other RNA-binding proteins.
Okay, so there’s a lot of jargon, but hopefully I can make the terms clear to understand!
- RbFox2= Fox2 is an RNA binding protein. It generally binds to a UGCAUG motif, and plays a crucial role in regulating alternative splicing.
- Alternative Splicing: When you transcribe your DNA to make mRNA, the mRNA consists of introns and exons. Introns are long non-coding sequences (they don’t contribute to making protein) that need to be spliced out of the transcript to create a contiguous sequence of exons. However, not all of the exons are always included in the final transcript, so alternative splicing is where you have different patterns of exons in the final transcript, because they have different levels of inclusion.
- Nonsense-Mediated Decay (NMD): Mature mRNA transcripts have a 5’ G-cap, 5’ UTR, coding region, 3’ UTR, 3’ poly-A tail, and a stop codon before the 3’UTR. When you have a premature stop codon (PTC), a stop codon that appears before it should, the mRNA transcript gets degraded instead of translated into truncated protein. This prevents the buildup of toxic proteins that are detrimental to the cell.
What I’m slaving at pipetting and tissue culture for <333- In detail
1) Sometimes, the transcripts produced from alternative splicing introduce a premature stop codon, which then undergo NMD= AS-NMD. Research contends that the presence of these transcripts don’t always represent “noise” in the alternative splicing process, but actually represent a functional process. It turns out that AS-NMD is used to regulate protein levels (especially for splicing factors like SR proteins/ hnRNPs), plays a role in development (you want to express certain proteins at certain times of development), and can be involved in negative feedback loops for protein expression. In summary, AS-NMD is an important post-transcriptional gene control mechanism, and naturally, its misregulation can lead to downstream issues.
It turns out that RbFox2 actually binds to these transcripts for other RNA-binding proteins! We hypothesize that it co-regulates the NMD process for these proteins, and we want to understand trends across different genes and tissues, and perhaps even uncover the mechanism by which it does so.
2) Chromatin describes the state of DNA interaction with various proteins (histones). The state of “openness” of the DNA is important because it affects what processes the DNA can undergo. The more open DNA is, the more able it is to undergo replication and transcription. Since splicing is mostly co-transcriptional (it actually happens at the same time as opposed to after transcription), we want to understand how different chromatin states affect alternative splicing patterns that are regulated by RbFox2. This is actually really important. Believe it or not, but adding methyl or acetyl groups to certain amino acid residues on histone proteins influences, or at least correlates with, different molecular processes and even disease phenotypes. The chromatin landscape is very dynamic and while the significance of many of the chromatin states are not well understood yet, you can bet there is a lot to uncover and understand the connections between those states and molecular biology processes that they affect.
3) Finally, I’m cloning histone methyltransferases! And I’m SO much better at cloning than I was last year!! These are enzymes that add methyl groups to lysine residues on specific histone proteins (details not so important), and our hope is to see how overexpression of these methyltransferases can affect alternative splicing mechanisms =)
And thus is a brief (it wasn’t very short though) summary of my research! It excites me SO much, and has been keeping me in lab for 10 hours a day, reading and re-reading papers and reviews to better understand the intricacies of what I’m investigating, and to really, appreciate the big picture of where my research fits into the larger network of questions and answers of molecular biology. I really love what I’m doing.
A pretty exciting thing is happening next week! My grad student Mohini is presenting her poster at the RNA Society Conference in Switzerland next week, and she’s featuring some of the data that I produced! And my name is on her poster… right next to Phil Sharp’s- just the association blows my mind ><
Anyways, I hope you guys followed/ enjoyed what I was saying, and if I was able to give you the tiniest appreciation for molecular biology, then I would say the past hour writing this was super worth it
Till more lab adventures next time!