Public Health Research Institute Center
UMDNJ - New Jersey Medical School
225 Warren Street
Newark, New Jersey 07103

Phone: (973) 854-3372
e-mail: tyagisa@umdnj.edu


Research Summary

Sanjay Tyagi and his colleagues have developed powerful probes to image single molecules of mRNAs in live and fixed cells. mRNA imaging is accomplished by using molecular beacons in live cells and by using single-molecule fluorescence in situ hybridization in fixed cells. They use these probes to study the dynamics of mRNA synthesis, processing, transport, and localization.

mRNA Traffic in Nucleus. For many years cell biologists have wondered 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. Tyagi and colleagues 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. With their ability to detect single mRNA molecules, they counted all mRNA molecules produced in individual cells by a gene. They 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 bursts in mRNA synthesis raises questions about how cells are able to maintain their relatively constant phenotypes in the face of such large-scale fluctuations.

Intracellular Venues of mRNA splicing. To image individual molecules of pre-mRNA and splicing products, two sets of probes were used at the same time, one set for an intron and the other set, labeled with a different color, for an exon. Usually splicing occurs at the gene locus, while the pre-mRNA is still being synthesized. However, with their single-molecule approach the laboratory 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 spliced in the nucleoplasm after their release from the gene locus.

Neuronal mRNA Transport. The group recently demonstrated that neuronal mRNAs travel into dendrites as the solitary cargo of the RNA transport granules. Their single-molecule imaging studies indicate that two molecules of the same or different mRNA species do not assemble in common structures. This model of mRNA transport affords a finer control of mRNA content within a synapse for synaptic plasticity then previous models did.

The current work is focused on understanding the mechanisms of mRNA transport and transcriptional bursts, and on imaging other mRNA processing events like non-sense mediated decay.






Selected Publications


Tyagi S, Kramer FR (2012) Molecular beacons in diagnostics. Faculty of 1000 Medicine Reports 4: 4-10.

Batish M, van den Bogaard P, Kramer FR, Tyagi S (2012) Neuronal mRNAs travel singly into dendrites. Proc Natl Acad Sci USA 109: 4645-4650. PMI: 22392993

Vargas DY, Shah K, Batish M, Levandoski M, Sinha S, Marras SA, Schedl P, Tyagi S (2011) Single-molecule imaging of transcriptionally coupled and uncoupled splicing. Cell 147: 1054-1065. PMI: 22118462

Tyagi S (2010) E. coli, what a noisy bug. Science 329: 518-519. PMI: 20671174

Tyagi S (2009) Imaging intracellular RNA distribution and dynamics in living cells. Nat Methods 6: 331-338. PMI: 19404252

Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5: 877-879. PMI: 18806792

Tyagi S (2007) RT-PCR enters the realm of stochastic gene expression. Genetic Engineering News: March 2007.

Tyagi S (2007) Splitting or stacking fluorescent proteins to visualize mRNA in living cells. Nat Methods 4: 391-392. PMI: 17464293

Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S (2006) Stochastic mRNA synthesis in mammalian cells. PLoS Biol 4: e309. PMI: 17048983

Vargas DY, Raj A, Marras SAE, Kramer FR, Tyagi S (2005) Mechanism of mRNA transport in the nucleus. Proc Natl Acad Sci USA 102: 17008-17013. PMI: 16284251

Mhlanga MM, Vargas DY, Fung CW, Kramer FR, Tyagi S (2005) tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells. Nucleic Acids Res 33: 1902-1912. PMI: 15809226

Tyagi S, Alsmadi O (2004) Imaging native beta-actin mRNA in motile fibroblasts. Biophys J 87: 4153-4162. PMI: 15377515

Bratu DP, Cha BJ, Mhlanga MM, Kramer FR, Tyagi S (2003) Visualizing the distribution and transport of mRNAs in living cells. Proc Natl Acad Sci USA 100: 13308-13313. PMI: 14583593

Marras SAE, Kramer FR, Tyagi S (2002) Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Research 30: e122. PMI: 12409481

Tyagi S, Marras SAE, Kramer FR (2000) Wavelength-shifting molecular beacons. Nature Biotechnology 18: 1191-1196.: PMI: 11062440

Marras SAE, Kramer FR, Tyagi S (1999) Multiplex detection of single-nucleotide variations using molecular beacons. Genetic Analysis: Biomolecular Engineering 14: 151-156.: PMI: 10084107

Bonnet G, Tyagi S, Libchaber A, Kramer FR (1999) Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc Natl Acad Sci USA 96: 6171-6176. PMI: 10339560

Tyagi S, Bratu DP, Kramer FR (1998) Multicolor molecular beacons for allele discrimination. Nat Biotechnol 16: 49-53. PMI: 9447593

Piatek AS, Tyagi S, Pol AC, Telenti A, Miller LP, Kramer FR, Alland D (1998) Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nat Biotechnol 16: 359-363. PMI: 9555727

Kostrikis LG, Tyagi S, Mhlanga MM, Ho DD, Kramer FR (1998) Spectral genotyping of human alleles. Science 279: 1228-1229. PMI: 9508692

Tyagi S, Landegren U, Tazi M, Lizardi PM, Kramer FR (1996) Extremely sensitive, background-free gene detection using binary probes and beta replicase. Proc Natl Acad Sci USA 93: 5395-5400. PMI: 8643586

Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14: 303-308. PMI: 9630890

Tyagi S, Ponnamperuma C (1990) Nonrandomness in prebiotic peptide synthesis. J Mol Evol 30: 391-399. PMI: 2111852

Lomeli H, Tyagi S, Pritchard CG, Lizardi PM, Kramer FR (1989) Quantitative assays based on the use of replicatable hybridization probes. Clin Chem 35: 1826-1831. PMI: 2673578

Tyagi S (1981) Origins of translation: the hypothesis of permanently attached adaptors. Orig Life 11: 343-351. PMI: 6799890



Awards


Jacob Heskel Gabbay Award in Biotechnology and Medicine, 2005.





C.V.


At Public Health Research Institute since 1987;
Ph.D. University of Maryland, 1987;
M.Phil. Jawaharlal Nehru University, 1982;
M.Sc. Jawaharlal Nehru University, 1980;
B.Sc. University of Rajasthan, 1978.