Research Summary

Sanjay Tyagi and his colleagues study the intracellular dynamics of messenger RNAs. To visualize the mRNAs in their living context, they have developed unique probes called molecular beacons that become fluorescent upon binding to specific sequences in messenger RNAs.

Illuminating oskar mRNA with these probes, they tracked its journey in developing Drosophila eggs. Oskar mRNA was seen to emerge from the nucleus of a nurse cell, funnel through the ring canal that connects the nurse cell with the growing oocyte, travel across the body of the oocyte, and finally settle at its posterior tip. Besides being the first visualization of a native mRNA in a living cell, this study uncovered the active transport mechanism that is responsible for oskar mRNA transport.

They studied the localization of B-actin mRNA in cells moving on flat surfaces. Time-lapse images of B-actin mRNA distribution in motile fibroblasts show that most of the actin mRNA is concentrated at the leading edges of the cells. At these locations, the cytoskeleton of the cells is in great turmoil - new actin fibers assemble from the monomeric actin, while old fibers dissolve. The high concentration of actin mRNA at the leading edges serves to provide a ready supply of fresh actin in this ferment.

Cell biologists have wondered for many years how the large complex formed by a mRNA and an entourage of proteins is able to travel from the site of transcription to the nuclear pores through the extremely dense nucleoplasm. Early theorists postulated active transport mechanisms, or proposed that transcriptionally active genes need to lie close to the periphery of the nucleus so that these mRNP complexes can exit from the pores located nearby. The laboratory was able to visualize the transport of individual mRNA molecules soon after their synthesis at gene loci in the nuclei of live cells. In order to detect single RNA molecules, they engineered an mRNA so that it is able to bind to multiple molecular beacons simultaneously, which renders the molecule so fluorescent that each mRNA molecule can be seen as a diffraction-limited spot in the microscope. The analysis of the tracks of individual molecules 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. The rate of mRNP diffusion is so fast that the mRNP complexes are dispersed throughout the nucleus soon after their synthesis and well before the onset of significant export into the cytoplasm. This glimpse into the dynamic architecture of the nucleus illuminates a key step in the expression of genes.

As a consequence of being able to detect single molecules, the laboratory counted all mRNA molecules produced in individual cells. They found that cells of higher eukaryotes display massive cell-to-cell variations in gene expression. Surpassing the magnitude of gene expression "noise" recently observed in prokaryotes and yeast, these variations stem from the nature of transcription in higher eukaryotes, which as their results demonstrate, occurs in bursts. Randomness in the onset and dissipation of mRNA synthesis, in combination with the short life time of mRNAs, causes significant variations in the amount of a given mRNA in each cell at any given moment. 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.




Recent Articles


Raj A, Peskin CS, Tranchina D, Vargas DY and Tyagi S (2006).
Stochastic mRNA synthesis in mammalian cells.
PloS Biology 4, October 2006; e309.

Vargas DV, Raj A, Marras SA, Kramer FR, Tyagi S. (2005).
Mechanism of mRNA transport in the nucleus.
Proc Natl Acad Sci USA. 2005 Nov 22;102(47):17008-13.

Mhlanga MM, Vargas DV, Fung CW, Kramer FR, Tyagi S. (2005).
tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells.
Nucleic Acids Res. 2005 April 4, 33(6):1902-12.

Tyagi S, Alsmadi OA. (2004).
Imaging native-beta-actin mRNA in motile fibroblasts.
Biophysical Journal, 2004 December, 87(6):4153-62.

Marras SA, Gold B, Kramer FR, Smith I, Tyagi S. (2004).
Real-time measurement of in vitro transcription.
Nucleic Acids Res. 2004 May 20;32(9):e72

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. 2003 Nov 11;100(23):13308-13.




Marras SA, Kramer FR, Tyagi S. (2002).
Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes.
Nucleic Acids Res 2002 Nov 1;30(21):e122
PMID: 12409481
Abstract  |  Full Article

Tyagi S, Marras SA, Kramer FR. (2000).
Wavelength-shifting molecular beacons.
Nat Biotechnol. 2000 Nov;18(11):1191-6
PMID: 11062440
Abstract  |  Full Article

Marras SA, Kramer FR, Tyagi S. (1999).
Multiplex detection of single-nucleotide variations using molecular beacons.
Genet Anal. 1999 Feb;14(5-6):151-6
PMID: 10084107
Abstract  |  Full Article

Bonnet G, Tyagi S, Libchaber A, Kramer FR. (1999).
Thermodynamic basis of the enhanced specificity of structured DNA probes.
Proc Natl Acad Sci U S A. 1999 May 25;96(11):6171-6
PMID: 10339560
Abstract  |  Full Article

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. 1998 Apr;16(4):359-63
PMID: 9555727
Abstract  |  Full Article

Kostrikis LG, Tyagi S, Mhlanga MM, Ho DD, Kramer FR. (1998).
Spectral genotyping of human alleles.
Science. 1998 Feb 20;279(5354):1228-9
PMID: 9508692
Abstract  |  Full Article

Tyagi S, Bratu DP, Kramer FR. (1998).
Multicolor molecular beacons for allele discrimination.
Nat Biotechnol. 1998 Jan;16(1):49-53
PMID: 9447593
Abstract  |  Full Article

Tyagi S, Kramer FR. (1996).
Molecular beacons: probes that fluoresce upon hybridization.
Nat Biotechnol. 1996 Mar;14(3):303-8
PMID: 9630890
Abstract

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 U S A. 1996 May 28;93(11):5395-400
PMID: 8643586
Abstract  |  Full Article

Tyagi S, Ponnamperuma C. (1990).
Nonrandomness in prebiotic peptide synthesis.
J Mol Evol. 1990 May;30(5):391-9
PMID: 2111852
Abstract

Lomeli H, Tyagi S, Pritchard CG, Lizardi PM, Kramer FR. (1989).
Quantitative assays based on the use of replicatable hybridization probes.
Clin Chem. 1989 Sep;35(9):1826-31
PMID: 2673578
Abstract

Tyagi S. (1981).
Origins of translation: the hypothesis of permanently attached adaptors.
Orig Life. 1981 Dec;11(4):343-51
PMID: 6799890
Abstract

PubMed Lisitings>




Research Grants


National Institutes of Health R01 Award
Visualizing the Movement of mRNAs in Living Cells
GM-070357; 2004 - 2008

National Institutes of Health R01 Award
Imaging the Transport of Individual mRNA Molecules to the Active Synapses
1R01MH079197; 2007 - 2011

National Institutes of Health R01 Award
High throughput PCR assays for diagnosing tuberculosis
1R03AI072105; 2007 - 2008


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, 1978;
B.Sc. University of Rajasthan, 1976.