Dr Manley's laboratory studies several aspects of gene expression in animal cells. These include transcription of mRNA encoding genes, mRNA splicing, and mRNA polyadenylation. All three of these processes 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 the complexes assemble and function, to learn how these important molecules act to regulate gene expression and how they themselves are controlled, and to understand how these processes contribute to cell growth, development and disease. These studies involve a large number of experimental approaches, including a variety of in vitro assays, biochemical fractionation and protein purification, structural analyses, and genetic studies using a variety of gene targeting approaches in yeast, mammalian cell culture and mice.

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 and members of the TLS/EWS family, function to link transcription and subsequent RNA processing, and how defects in these factors contribute to human disease including cancer. We are also interested in the transcribing enzyme itself, RNA polymerase II, and especially interactions with factors that allow it to elongate efficiently and eventually to terminate transcription, a remarkably complex process.

Our studies on mRNA splicing address a number of different issues. We study the mechanism of pre-mRNA splicing by examining the role that small nuclear RNAs (snRNAs) play in the catalysis of splicing. For example, we showed for the first time that fragments of two of these snRNAs, U2 and U6, can by themselves catalyze reactions that resemble the authentic splicing reaction. We are also very interested in regulation of alternative splicing, an important mechanism of gene control. We concentrate on understanding how members of the SR protein family, characterized by a human protein we co-discovered called ASF/SF2, function to modulate the selection of splice sites in alternatively spliced pre-mRNAs and to control splicing more generally and under a variety of physiological conditions; how these proteins are regulated by posttranslational modifications such as phosphorylation; and how these and related proteins contribute to cancer, heart disease and neurological conditions.

Addition of the poly(A) tail to an mRNA is the last step in the synthesis of mRNA, and it, too, is a highly regulated process that requires numerous protein factors. These factors, including the poly(A) polymerase itself, constitute an interesting family of proteins that interact with each other and with the mRNA precursor in novel ways. 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 also uncovered interactions with DNA repair factors and tumor suppressor proteins, which suggest an unexpected interplay between these nuclear processes, and obtained evidence that the process is regulated extensively by protein sumoylation.
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 contribute to gene control.