Regulation of mRNA synthesis in animal cells.
Dr. Manley's laboratory studies several aspects of gene expression, principally in human cells. These include transcription of mRNA encoding genes, and splicing and polyadenylation of the resultant mRNA precursors. These processes all occur in the cell nucleus and require numerous protein (and in the case of splicing, RNA) factors that assemble into massive multi-subunit complexes. Important goals are to understand how these molecules act to regulate gene expression and how they themselves are controlled, and to understand how these processes contribute to cell growth and development, and to disease when dysregulated. These studies involve a large number of experimental approaches, including a variety of in vitro assays, biochemical fractionation and protein purification, structural analyses, genetic studies using a variety of gene targeting approaches in yeast and mammalian cells, and analysis of gene expression in human cell lines and pathological samples.
With respect to transcription, we are studying several factors that function in gene control. This includes analyzing both biochemically and genetically how certain transcription factors, such as the evolutionarily conserved PAF complex, the RNA/DNA helicase Senataxin and the RNA/DNA binding protein FUS, function to link transcription and subsequent RNA processing, and how mutations in these factors contribute to human diseases, especially neurodegenerative diseases such as ALS, or Lou Gehrig's disease.
Our studies on mRNA splicing address a number of different issues. We are very interested in regulation of alternative splicing, an important mechanism of gene control. We concentrate on understanding how RNA binding proteins, including members of the hnRNP and SR protein families, function to modulate the selection of splice sites in alternatively spliced pre-mRNAs and to control splicing more generally under a variety of physiological conditions, such as the maintenance of pluripotency and subsequent differentiation of human embryonic stem cells; and how these and related proteins, when misexpressed or mutated, contribute to cancer and neurodegenerative disease. Along these lines, we also study how mutations in genes encoding subunits of the core splicing machinery, or spliceosome, lead to specific defects in alternative splicing and cause diseases such as myelodysplastic syndromes and a growing number of cancers.
Addition of the poly(A) tail to an mRNA precursor, which involves endonucleolytic cleavage and synthesis of a poly(A) tail, is the last step in the synthesis of mRNA, and it, too, is a highly regulated process that requires numerous protein factors. Several of these proteins play important regulatory roles in different cell types, during cell differentiation, and at different stages of the cell cycle. Our studies have uncovered interactions with DNA repair factors and tumor suppressor proteins, which suggest an unexpected interplay between these nuclear processes, and have also provided evidence that these interactions are regulated extensively by protein sumoylation. Finally, like alternative splicing, alternative polyadenylation, which is the selection of distinct sites of cleavage and polyadenylation in the pre-mRNA, is now known to be a widespread mechanism of gene control, and we are studying its regulation during cell differentiation and disease.
Our lab and others have shown that these three processes, transcription, splicing and polyadenylation, are all linked, or coupled, in interesting ways. For example, RNA polymerase II, in addition to its role in transcription, also functions directly in both splicing and polyadenylation. This requires a unique region of the polymerase, known as the CTD, which consists of a long, repetitive sequence that is highly phosphorylated. We are currently studying how the CTD functions, and how its interactions with other proteins contribute to gene control.
- Zhang, J., Lieu, Y.K., Ali, A.M., Penson, C., Reggio, K.S., Rabadan, R., Raza, A., Mukherjee, S. and Manley, J.L. (2015). A disease-associated mutation in SRSF2 misregulates splicing by altering RNA binding affinities. Proc. Natl. Acad. Sci. USA. 112, E4726-34.
- Conlon, E.G., Lu, L., Sharma, A., Yamazaki, T., Tang, T., Shneider, N.A. and Manley, J.L. (2016). The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS patient brains. eLife 5, e17820.
- Ogami, K., Richard, P., Chen, Y., Hoque, M., Li, W., Moresco, J.J., Yates III, J.R., Tian, B. and Manley, J.L. (2017). An Mtr4/ ZFC3H1 complex facilitates turnover of unstable nuclear RNAs to prevent their cytoplasmic transport and global translational repression. Genes Dev. 31, 1257-1271.
- Yurko, N., Liu, X., Yamazaki, T., Hoque, M., Tian, B. and Manley, J.L. (2017). MPK1/SLT2 links multiple stress responses with gene expression in budding yeast by phosphorylating Tyr1 of the RNAP II CTD. Mol. Cell. 68, 913-925.
- Conlon, E.G., Fagegaltier, D., Agius, P., Davis-Porada, J., Gregory, J., Hubbard, I. Kang, K., Kim, D., The NYGC ALS Consortium, Phatnani, H., Shneider, N.A. and Manley, J.L. (2018). Unexpected similarities between C9ORF72 and sporadic forms of ALS/FTD suggest a common disease mechanism. eLife 7, e37754.
- Yamazaki, T., Liu, L., Lazarev, D., Al-Zain, A., Fomin, V., Yeung, P.L., Chambers, S.M., Lu, C.W., Studer, L. and Manley, J.L. (2018). TCF3 alternative splicing controlled by hnRNP H/F regulates E-cadherin expression and hESC pluripotency. Genes Dev. 32, 1161-1174.
- Zhang, J., Ali, A.M., Lieu, Y.K., Liu, Z., Gao, J., Rabadan, R., Raza, A., Mukherjee, S. and Manley, J.L. (2019). Disease-causing mutations in SF3B1 alter splicing by disrupting interaction with SUGP1. Mol. Cell 76, 82-95.
- Tsai, Y.L., Coady, T.H., Lu, L., Zheng, D., Alland, I., Tian, B., Shneider, N.A. and Manley, J.L. (2020). ALS/FTD-associated protein FUS induces mitochondrial dysfunction by preferentially sequestering respiratory chain complex mRNAs. Genes Dev. 34, 785-805.