©2018 by Sanjay Tyagi Lab. Proudly created with Wix.com

SANJAY TYAGI LAB

Exploring RNA Biology through Imaging Single Molecules in situ

 

OUR RESEARCH

 

mRNA Traffic in Nucleus

How the large complex formed by mRNA and the proteins that bind to it during its synthesis, is able to travel from the site of transcription to the nuclear pores through the extremely dense chromatin has been an enigma. We visualized the transport of individual mRNA molecules soon after their synthesis at the gene locus. The analysis of the molecular tracks revealed that mRNP complexes explore the volume of the nucleus by simple Brownian diffusion. The motion of mRNP complexes is restricted to the interchromatin spaces. When the complexes drift into the dense chromatin, they tend to become immobilized. This glimpse into the dynamic architecture of the nucleus reveals a key step in the expression of genes.

Stochastic mRNA Synthesis

Using single molecule FISH we counted mRNA molecules produced in individual cells by a gene. We found that cells of higher eukaryotes display massive cell-to-cell variations in gene expression. The variations occur because the mRNAs are produced in randomly initiated bursts and then decay in a steady manner.  The observation of these cell-to-cell variations raises questions about how cells are able to maintain their relatively constant phenotypes in the face of such large-scale variations.  Current work in the laboratory is focused on the mechanisms that renders transcription intermittent rather than steady.

Intracellular Venues of mRNA splicing

Most introns are removed rapidly while the pre-mRNA is still being synthesized at the gene locus.  However, during alternative splicing the splicing decisions must be differed until all the introns involved in the multiple choice are synthesized.  To image individual molecules of pre-mRNA and splicing products, we used two sets of single-molecule FISH probes at the same time, one set for an intron and the other set, labeled with a different color, for an exon.  We found that during alternative splicing regulated by RNA binding proteins, the normally tight coupling of transcription and splicing is broken, and the pre-mRNAs are released from the gene locus.  These pre-mRNA molecules then reach to the periphery of “speckles”, the nuclear bodies where splicing factors are concentrated, to complete the final event of splicing.

Traveling solo to the distant reaches of neurons

Brain stores memory by forming unique connections between neurons called synapses and by differentiating synapses that are used repeatedly.  To aid in the process of differentiation a number of mRNA species are enriched within dendrites at such synapses and are locally translated.  These mRNAs arrive at these sites as a cargo of large RNA-protein complexes called RNA transport granules.  We imaged pairwise combinations of a number of these mRNAs with a single-molecule resolution and found that two molecules are same or different species are never co-localized.  This indicates that mRNA molecules traffic to the distal reaches of dendrites singly and independently of others and not in clusters.

OUR PROBES

 

Molecular Beacons

Molecular beacons are probes that become fluorescent when they recognize and bind to a complementary DNA or RNA.  Shaped like a hairpin, they are made from synthetic pieces of DNA with a pair of fluorescent and quencher dyes attached at their termini.  In the hairpin conformation of the probe, the fluorescence of the fluorophore is quenched but it is restored when the loop binds to the complementary target.  These probes allow monitoring of progress reactions that produce specific nucleic acids, such as polymerase chain reactions, in sealed tubes and real time.  They can also be used to detect and image mRNAs in live cells using a fluorescence microscope.  Such studies allow us to explore the dynamics of gene expression and RNA localization.

Single-Molecule FISH

We use about 50-short fluorescently labeled probes to bind to the same mRNA in fixed cells.  Deposition of so many probes on the same target renders mRNA molecules so intensely fluorescent that they becomes visible as  bright spots in a microscope.  Since the spots arises from the combined binding of all or most of the probes, only the intended targets can create a spot like signals.  The number of spots accurately reflects the number of mRNA molecules in the cell at the time of fixation.  By labeling two segments of the same mRNA with distinctly labeled probe sets, it is possible to study events that segregate the parts (splicing), or bring them together (gene fusion).   Prelabeled sets of probes for smFISH are available commercially as Stellaris probes from Biosearch LGC.

bottle2.jpg
 

Our laboratory is a part of Public Health Research Institute, New Jersey Medical School, Rutgers University. 
It is located in the ICPH Building 225 Warren Street Newark, NJ 07103, United States

Screen Shot 2018-10-11 at 11.40.01 PM co
RU_SIG_NJMS_CMYK_K.jpg