Shalini Sharma

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Courses

Scholarly Contributions

Chapters

• Wongpalee, S., & Sharma, S. (2014). The pre-mRNA splicing reaction.. In Methods in Molecular Biology series- Spliceosomal Pre-mRNA Splicing(pp 3-12). Humana press.
• Sharma, S. (2008). Isolation of polypyrimidine tract binding protein using RNA affinity chromatography. In RNA-Protein Interaction Protocols, second edition. Humana press.

Journals/Publications

• Schippel, N., & Sharma, S. (2023). Dynamics of human hematopoietic stem and progenitor cell differentiation to the erythroid lineage. Experimental Hematology, 123, 1-17.
• Wong, J., Martelly, W., & Sharma, S. (2022). Sequence-specific RNA recognition by an RGG motif connects U1 and U2 snRNP for spliceosome assembly. PNAS.
• Bapat, A., Schippel, N., Shi, X., Jasbi, P., Gu, H., Kala, M., Sertil, A., & Sharma, S. (2021). Hypoxia promotes erythroid differentiation through the development of progenitors and proerythroblasts. Experimental Hematology.
• Martelly, W., Fellows, B., Kang, P., Vashisht, A., Wohlschlegel, J., & Sharma, S. (2021). Synergistic actions of U1 snRNA stem-loops in pre-mRNA splicing. RNA Biology.
• Wong, J., Martelly, W., & Sharma, S. (2021). A reporter based cellular assay for monitoring splicing efficiency. Journal of Visualized Experiments.
• Bapat, A., Keita, N., & Sharma, S. (2019). Pan-myeloid differentiation of human cord blood derived CD34+ hematopoietic stem and progenitor cells. Journal of Visualized Experiments, 150, e59836.
• Martelly, W., Fellows, B., Senior, K., Marlowe, T., & Sharma, S. (2019). Identification of a non-canonical RNA binding domain in the U2 snRNP protein SF3A1. RNA, 25, 1509-1521.
• Bapat, A., Keita, N., Martelly, W., Kang, P., Seet, C., Jacobsen, J. R., Stoilov, P., Hu, C., Crooks, G. M., & Sharma, S. (2018). Myeloid Disease Mutations of Splicing Factor SRSF2 Cause G2-M Arrest and Skewed Differentiation of Human Hematopoietic Stem and Progenitor Cells. Stem cells (Dayton, Ohio), 36(11), 1663-1675.
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  Myeloid malignancies, including myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia, are characterized by abnormal proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). Reports on analysis of bone marrow samples from patients have revealed a high incidence of mutations in splicing factors in early stem and progenitor cell clones, but the mechanisms underlying transformation of HSPCs harboring these mutations remain unknown. Using ex vivo cultures of primary human CD34 cells as a model, we find that mutations in splicing factors SRSF2 and U2AF1 exert distinct effects on proliferation and differentiation of HSPCs. SRSF2 mutations cause a dramatic inhibition of proliferation via a G2-M phase arrest and induction of apoptosis. U2AF1 mutations, conversely, do not significantly affect proliferation. Mutations in both SRSF2 and U2AF1 cause abnormal differentiation by skewing granulo-monocytic differentiation toward monocytes but elicit diverse effects on megakaryo-erythroid differentiation. The SRSF2 mutations skew differentiation toward megakaryocytes whereas U2AF1 mutations cause an increase in the erythroid cell populations. These distinct functional consequences indicate that SRSF2 and U2AF1 mutations have cell context-specific effects and that the generation of myeloid disease phenotype by mutations in the genes coding these two proteins likely involves different intracellular mechanisms. Stem Cells 2018;36:1663-1675.
• Keppetipola, N. M., Yeom, K., Hernandez, A. L., Bui, T., Sharma, S., & Black, D. L. (2016). Multiple determinants of splicing repression activity in the polypyrimidine tract binding proteins, PTBP1 and PTBP2. RNA (New York, N.Y.), 22(8), 1172-80.
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  Most human genes generate multiple protein isoforms through alternative pre-mRNA splicing, but the mechanisms controlling alternative splicing choices by RNA binding proteins are not well understood. These proteins can have multiple paralogs expressed in different cell types and exhibiting different splicing activities on target exons. We examined the paralogous polypyrimidine tract binding proteins PTBP1 and PTBP2 to understand how PTBP1 can exhibit greater splicing repression activity on certain exons. Using both an in vivo coexpression assay and an in vitro splicing assay, we show that PTBP1 is more repressive than PTBP2 per unit protein on a target exon. Constructing chimeras of PTBP1 and 2 to determine amino acid features that contribute to their differential activity, we find that multiple segments of PTBP1 increase the repressive activity of PTBP2. Notably, when either RRM1 of PTBP2 or the linker peptide separating RRM2 and RRM3 are replaced with the equivalent PTBP1 sequences, the resulting chimeras are highly active for splicing repression. These segments are distinct from the known region of interaction for the PTBP1 cofactors Raver1 and Matrin3 in RRM2. We find that RRM2 of PTBP1 also increases the repression activity of an otherwise PTBP2 sequence, and that this is potentially explained by stronger binding by Raver1. These results indicate that multiple features over the length of the two proteins affect their ability to repress an exon.
• Vashisht, A. A., Sharma, S., Chui, D., Wohlschlegel, J. A., Black, D. L., & Wongpalee, S. P. (2016). Author response: Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing. eLife. doi:10.7554/elife.19743.014
• Wongpalee, S. P., Vashisht, A., Sharma, S., Chui, D., Wohlschlegel, J. A., & Black, D. L. (2016). Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing. eLife, 5.
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  Polypyrimidine-tract binding protein PTBP1 can repress splicing during the exon definition phase of spliceosome assembly, but the assembly steps leading to an exon definition complex (EDC) and how PTBP1 might modulate them are not clear. We found that PTBP1 binding in the flanking introns allowed normal U2AF and U1 snRNP binding to the target exon splice sites but blocked U2 snRNP assembly in HeLa nuclear extract. Characterizing a purified PTBP1-repressed complex, as well as an active early complex and the final EDC by SILAC-MS, we identified extensive PTBP1-modulated changes in exon RNP composition. The active early complex formed in the absence of PTBP1 proceeded to assemble an EDC with the eviction of hnRNP proteins, the late recruitment of SR proteins, and binding of the U2 snRNP. These results demonstrate that during early stages of splicing, exon RNP complexes are highly dynamic with many proteins failing to bind during PTBP1 arrest.
• Sharma, S. (2014). Stem-loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly. Genes and Development, 28(22), 2518-31.
• Sharma, S. (2014). U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro. Gene, 540(1), 11-15.
• Keppetipola, N., Sharma, S., Li, Q., & Black, D. L. (2013). Neuronal regulation of pre-mRNA splicing by polypyrimidine tract binding proteins, PTBP1 and PTBP2. Critical reviews in biochemistry and molecular biology, 47(4).
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  Alternative splicing patterns are regulated by RNA binding proteins that assemble onto each pre-mRNA to form a complex RNP structure. The polypyrimidine tract binding protein, PTB, has served as an informative model for understanding how RNA binding proteins affect spliceosome assembly and how changes in the expression of these proteins can control complex programs of splicing in tissues. In this review, we describe the mechanisms of splicing regulation by PTB and its function, along with its paralog PTBP2, in neuronal development.
• Sharma, S., Maris, C., Allain, F. H., & Black, D. L. (2011). U1 snRNA directly interacts with polypyrimidine tract-binding protein during splicing repression. Molecular cell, 41(5).
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  Splicing of the c-src N1 exon is repressed by the polypyrimidine tract-binding protein (PTB or PTBP1). During exon repression, the U1 snRNP binds properly to the N1 exon 5' splice site but is made inactive by the presence of PTB. Examining the patterns of nuclease protection at this 5' splice site, we find that the interaction of U1 is altered by the adjacent PTB. Interestingly, UV crosslinking identifies a direct contact between the pre-mRNA-bound PTB and the U1 snRNA. EMSA, ITC, and NMR studies show that PTB RRMs 1 and 2 bind the pyrimidine-rich internal loop of U1 snRNA stem loop 4. The PTB/U1 interaction prevents further assembly of the U1 snRNP with spliceosomal components downstream. This precise interaction between a splicing regulator and an snRNA component of the spliceosome points to a range of different mechanisms for splicing regulation.
• Tang, Z. Z., Sharma, S., Zheng, S., Chawla, G., Nikolic, J., & Black, D. L. (2011). Regulation of the mutually exclusive exons 8a and 8 in the CaV1.2 calcium channel transcript by polypyrimidine tract-binding protein. The Journal of biological chemistry, 286(12).
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  CaV1.2 calcium channels play roles in diverse cellular processes such as gene regulation, muscle contraction, and membrane excitation and are diversified in their activity through extensive alternative splicing of the CaV1.2 mRNA. The mutually exclusive exons 8a and 8 encode alternate forms of transmembrane segment 6 (IS6) in channel domain 1. The human genetic disorder Timothy syndrome is caused by mutations in either of these two CaV1.2 exons, resulting in disrupted Ca(2+) homeostasis and severe pleiotropic disease phenotypes. The tissue-specific pattern of exon 8/8a splicing leads to differences in symptoms between patients with exon 8 or 8a mutations. Elucidating the mechanisms controlling the exon 8/8a splicing choice will be important in understanding the spectrum of defects associated with the disease. We found that the polypyrimidine tract-binding protein (PTB) mediates a switch from exon 8 to 8a splicing. PTB and its neuronal homolog, nPTB, are widely studied splicing regulators controlling large sets of alternative exons. During neuronal development, PTB expression is down-regulated with a concurrent increase in nPTB expression. Exon 8a is largely repressed in embryonic mouse brain but is progressively induced during neuronal differentiation as PTB is depleted. This splicing repression is mediated by the direct binding of PTB to sequence elements upstream of exon 8a. The nPTB protein is a weaker repressor of exon 8a, resulting in a shift in exon choice when nPTB replaces PTB in cells. These results provide mechanistic understanding of how these two exons, important for human disease, are controlled.
• Babic, I., Sharma, S., & Black, D. L. (2009). A role for polypyrimidine tract binding protein in the establishment of focal adhesions. Molecular and cellular biology, 29(20).
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  Polypyrimidine tract binding protein (PTB) is a widely expressed RNA binding protein. In the nucleus PTB regulates the splicing of alternative exons, while in the cytoplasm it can affect mRNA stability, translation, and localization. Here we demonstrate that PTB transiently localizes to the cytoplasm and to protrusions in the cellular edge of mouse embryo fibroblasts during adhesion to fibronectin and the early stages of cell spreading. This cytoplasmic PTB is associated with transcripts encoding the focal adhesion scaffolding proteins vinculin and alpha-actinin 4. We demonstrate that vinculin mRNA colocalizes with PTB to cytoplasmic protrusions and that PTB depletion reduces vinculin mRNA at the cellular edge and limits the size of focal adhesions. The loss of PTB also alters cell morphology and limits the ability of cells to spread after adhesion. These data indicate that during the initial stages of cell adhesion, PTB shuttles from the nucleus to the cytoplasm and influences focal adhesion formation through coordinated control of scaffolding protein mRNAs.
• Sharma, S. (2008).

  Isolation of a sequence-specific RNA binding protein, polypyrimidine tract binding protein, using RNA affinity chromatography.

  . Methods in molecular biology (Clifton, N.J.), 488, 1-8. doi:10.1007/978-1-60327-475-3_1
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  Many important cellular processes are mediated by sequence-specific RNA binding proteins, and it is often necessary to purify these proteins. When the RNA binding site is known, it is convenient to use this RNA as a matrix for affinity purification. The intronic splicing silencer (ISS) element present upstream of the N1 exon of the c-src pre-mRNA is a high-affinity binding site for the polypyrimidine tract binding protein (PTB). Using a 5'-biotinylated ISS RNA and PTB as an example, I describe a one-step method for affinity chromatography of RNA binding proteins from nuclear extracts.
• Sharma, S., Kohlstaedt, L., Damianov, A., Rio, D. C., & Black, D. L. (2008). Polypyrimidine tract binding protein controls transition from exon definition to an intron defined spliceosome. Nat. Struct. Mol. Biol., 183-191.
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  (Highlighted in Schellenberg et. al (2008) TIBS 33, 243-246)
• Sharma, S., & Black, D. L. (2006). Maps, Codes, and Sequence Elements: Can We Predict the Protein Output from an Alternatively Spliced Locus?. Neuron, 574-576.
• Chowdhury, A. R., Sharma, S., Mandal, S., Goswami, A., Mukhopadhyay, S., & Majumder, H. K. (2002). Luteolin, an emerging anti-cancer flavonoid, poisons eukaryotic DNA topoisomerase I. Biochemical J., 653-661.
• Dasgupta, A., Sharma, S., Das, A., Sarkar, D., & Majumder, H. K. (2002). Carboxy-terminal domain of the largest subunit of RNA pol II of Leishmania donovani has an unusually low number of phosphorylation sites. Medical Sci. Monit., 341-350.
• Das, A., Dasgupta, A., Sharma, S., Ghosh, M., Sengupta, T., Bandhopadhyay, S., & Majumder, H. K. (2001). Characterization of the gene encoding type II DNA topoisomerase from Leishmania donovani: a key molecular target in antileishmanial therapy. Nucl. Acids Res., 1844-1851.
• Sharma, S. (2001). Comparative studies on kinetics of inhibition of protein synthesis in intact cells by ricin and a conjugate of ricin B-chain with momordin. Mol. Cell. Biochem..

Presentations

• Sharma, S. (2023, May 25). Splicing regulation and its deregulation in myeloid diseases (Invited Seminar). Center for RNA Biology and Therapeutics, City of Hope, California.
• Sharma, S., & Martelly, W. (2017, August). RNA helicase UAP56 mediates early splice site pairing by promoting contact between U1 and U2 snRNP. 11th Cold Spring Harbor Laboratory Meeting: Eukaryotic mRNA Processing, Long Island, NY (August 22-26, 2017). Long Island, NY.
• Sharma, S., Bapat, A., & Keita, N. (2017, May). Understanding the role of splicing factor mutation in myelodysplastic syndromes. 1st Arizona RNA Salon Symposium, University of Arizona, Tucson, AZ (May 19, 2017). Tucson, AZ.
• Sharma, S. (2012, April). A role for the stemloop 4 of U1 snRNA in pre-mRNA splicing. Annual Meeting of the American Society of Biochemistry and Molecular Biology. San Diego, California.
• Sharma, S. (2012, Spring). A role for the stem loop 4 of U1 snRNA in splice site pairing.. Seventeenth Annual Meeting of the RNA Society. Ann Arbor, Michigan.
• Sharma, S. (2009, August). PTB interacts with 5 splice site bound U1 snRNP during splicing repression. Eukaryotic mRNA Processing Meeting. Cold Spring Harbor Laboratory, New York.
• Sharma, S. (2007, June). The transition from an exon definition complex to an intron defined spliceosome is blocked by the polypyrimidine tract binding protein.. Twelfth Annual Meeting of the RNA Society,. Madison, Wisconsin.

Poster Presentations

• Schippel, N., & Sharma, S. (2023, November). Single Cell Analysis of Human Early Erythroid Progenitor Heterogeneity. Single Cell Analysis Meeting at the Cold Spring harbor Laboratory, Long Island, NY (November 8-11, 2023). Long Island, NY.
• Schippel, N., Sharma, S., Schippel, N., & Sharma, S. (2023, April 28). Cytokine Dependence of Population Dynamics in Human Erythropoiesis. 8th Annual Arizona Biomedical Research Commission - Flinn Research Conference.
• Wong, J., Yellamaty, R., Martelly, W., & Sharma, S. (2023, May 31-June 4). Splicing Activity of Human Variant U1 snRNAs. Annual Meeting of the RNA Society.
• Sharma, S. (2022, May1-4). Splicing functions of the U1 snRNA – Beyond 5’ Splice Site Recognition. Keystone Symposia on Molecular and Cellular Biology: Small Regulatory RNAs- From Bench to Bedside.
• Sharma, S., & Schippel, N. (2022, May 31-June 4). Cytokine Dependence of Population Dynamics in Human Erythropoiesis. Annual Meeting of the RNA Society.
• Sharma, S., & Schippel, N. (2022, October 28). Cytokine Dependence of Population Dynamics in Human Erythropoiesis. Annual Retreat of the University of Arizona Cancer Center.
• Sharma, S., & Schippel, N. (2022, September 1-4). Cytokine Dependence of Population Dynamics in Human Erythropoiesis. Annual Meeting of the International Society of Experimental Hematology.
• Sharma, S., & Smith, R. (2022, October 28). Differential SCF Signaling Under Hypoxia-Induced Stress in Erythroid Progenitor K-562 Cells. Annual Retreat of the University of Arizona Cancer Center.
• Sharma, S., & Yellamaty, R. (2022, May 31-June 4). Determinants of selective binding to U1-SL3 by the DExD-box helicase UAP56. Annual Meeting of the RNA Society.
• Sharma, S., & Yellamaty, R. (2022, October 28). Determinants of selective binding to U1-SL3 by the DExD-box helicase UAP56. Annual Retreat of the University of Arizona Cancer Center.
• Sharma, S. (2019, August). Stem-loops 3 and 4 of the U1 snRNA have synergistic roles in splicing. 12th Cold Spring Harbor Laboratory Meeting: Eukaryotic mRNA Processing, Long Island, NY (August 21-24, 2019).
• Sharma, S. (2019, February 24-28). A non-canonical RNA-binding domain in SF3A1. Keystone Symposia on RNA-Protein Interactions. Whistler, British Columbia.
• Sharma, S., Martelly, W., & Fellows, B. (2019, August 9th). A non-canonical RNA-binding domain in SF3A1. 3rd Annual Arizona RNA Salon Symposium. Tucson, AZ.
• Sharma, S., Fellows, B., & Martelly, W. (2018, 2018-11-30). Splicing factor 3A1 interacts with the stem-loop 4 of U1 snRNA via its ubiquitin like domain.. Fall 2018 Student Colloquium of the Community College Undergraduate Research Initiative. Glendale Community College: The Community College Undergraduate Research Initiative.
• Sharma, S., Martelly, W., & Fellows, B. (2018, May 14th). Estimating the genome-wide prevalence of SF3A1-dependent splice site pairing. 2nd Annual Arizona RNA Salon Symposium. Phoenix, AZ.
• Sharma, S., Martelly, W., & Fellows, B. (2018, May 14th). Investigating the interactions of splicing factor SF3A1 with the stem-loop 4 of U1 snRNA and RNA helicase UAP56. 2nd Annual Arizona RNA Salon Symposium. Berkeley, CA.
• Sharma, S., Martelly, W., & Fellows, B. (2018, May 29th-June 3rd). Estimating the genome-wide prevalence of SF3A1-dependent splice site pairing. 23rd Annual Meeting of the RNA Society. Berkeley, CA.
• Sharma, S., Martelly, W., & Fellows, B. (2018, May 29th-June 3rd). Investigating the interactions of splicing factor SF3A1 with the stem-loop 4 of U1 snRNA and RNA helicase UAP56. 23rd Annual Meeting of the RNA Society. Berkeley, CA.
• Sharma, S. (2017, August). Effects of splicing factor mutations on human hematopoietic stem and progenitor cells.. 11th Cold Spring Harbor Laboratory Meeting: Eukaryotic mRNA Processing, Long Island, NY (August 22-26, 2017). Cold Spring Harbor Laboratory, New York.
• Sharma, S. (2017, March). Effects of splicing factor mutations on hematopoietic stem and progenitor cells.. Keystone Symposia on mRNA Processing and Human Disease. Taos, New Mexico.
• Sharma, S., & Keita, N. (2017, August). Effect of inhibitors of CLK and SRPK family kinases on the phosphorylation state of SRSF2 and the survival of several AML cell lines and CD34+ HSPCs. 11th Cold Spring Harbor Laboratory Meeting: Eukaryotic mRNA Processing, Long Island, NY (August 22-26, 2017). Long Island, NY.
• Sharma, S., & Martelly, W. (2017, May). RNA helicase UAP56 mediates early splice site pairing by promoting contact between U1 and U2 snRNP. 1st Arizona RNA Salon Symposium, University of Arizona, Tucson, AZ (May 19, 2017). Tucson, AZ.
• Sharma, S., & Martelly, W. (2017, October). RNA helicase UAP56 mediates early splice site pairing by promoting contact between U1 and U2 snRNP. AZBio Awards, Arizona BioIndustry Association, Phoenix, AZ, (October 11, 2017). Phoenix, AZ.
• Sharma, S., & Martelly, W. (2017, October). RNA helicase UAP56 mediates early splice site pairing by promoting contact between U1 and U2 snRNP. Molecular and Cellular Biology/Microbiology Graduate Programs Retreat, Arizona State University, Tempe, AZ (October 10, 2017). Phoenix, AZ.
• Sharma, S., Bapat, A., & Keita, N. (2017, May). Morphological characterization of human CD34+ cells expressing MDS-associated mutations of SRSF2.. 3rd Annual University of Arizona Cancer Center Retreat, Tucson, AZ (April 21, 2017). Tucson, AZ.
• Sharma, S., Bapat, A., & Keita, N. (2017, May). Understanding the role of splicing factor mutation in myelodysplastic syndromes. 3rd Annual University of Arizona Cancer Center Retreat, Tucson, AZ (April 21, 2017). Tucson, AZ.
• Sharma, S. (2014, June). Stemloop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP specific SF3A1 protein during spliceosome assembly.. Nineteenth Annual Meeting of the RNA Society. Quebec City, Canada.
• Sharma, S. (2014, October). Role of splicing factor mutations in development of myelodysplastic syndromes. Cancer Biology Retreat- University of Arizona Cancer Center- October 4th. Tucson.
• Sharma, S. (2013, August). Effect of MDS mutations on splicing factor function. Eukaryotic mRNA Processing Meeting,. Cold Spring Harbor Laboratory, New York.
• Sharma, S. (2007, August). Polypyrimidine tract binding protein blocks the transition from an exon definition complex to an intron defined spliceosome.. Eukaryotic mRNA Processing Meeting. Cold Spring Harbor Laboratory, New York.
• Sharma, S. (2006, June). Polypyrimidine tract binding protein interacts with the exon bound U1 snRNP during splicing repression.. Eleventh Annual Meeting of the RNA Society. Seattle Washington.
• Sharma, S. (2005, December). Polypyrimidine tract binding protein blocks the 5 splice site dependent assembly of U2AF and the prespliceosomal E complex.. The American Society for Cell Biology 45th Annual Meeting. San Francisco California.
• Sharma, S. (2004, June). Pre-spliceosomal complexes of the c-src pre-mRNA.. Ninth Annual Meeting of the RNA Society. Madison, Wisconsin.
• Sharma, S. (2002, June). RNP-complexes of the c-src Pre-mRNA.. Seventh Annual Meeting of the RNA Society. Madison, Wisconsin.