Highlights d Cryo-EM structure of the human spliceosomal C complex at 3.4 A ˚core resolution d Localization and likely function of novel C-specific proteins in human C complexes d Metazoan-specific proteins stabilize key protein and RNA regions in human C complexes d The timing of RNP rearrangements differs in human and yeast C/C * complexes

The discovery of constituents of eukaryotic transcription systems was enabled by the development of in vivo and in vitro methods for their study. Depending on the organism to be studied, the relative importance of each of these two general approaches has been different. Our knowledge about transcription of human genes would be considerably smaller without the results that were obtained by employing in vitro methods. Here, we will mainly focus on in vitro studies of gene expression that contributed to discoveries in the human RNA polymerase (RNAP) III transcription system.In contrast to unicellular yeast, it has until today been and will most likely remain for a many more years impossible to study human RNAP III transcription in cells that have been grown under conditions resembling at least approximately to an in vivo situation in a human being. For that reason, it has hitherto been difficult to address questions concerning the physiological regulation of human RNAP III transcription in its natural environment. However, the identification of the basal components of the RNAP III transcription system could be achieved without directly resorting to a human "in vivo model system." Yet, the identification of human RNAP III subunits and of accessory transcription factors repeatedly took advantage of in vivo systems that were established in other species. Many discoveries of components of the human RNAP III transcription system were fostered by work performed in unicellular (for example, S. cerevisiae and S. pombe) and multicellular eukaryotes (among others, X. laevis or D. melanogaster). Often, results obtained in these model systems helped researchers identify proteins of the human RNAP III transcription system. For instance, the amino acid sequences of transcription factors that were identified and cloned by employing in vivo and in vitro methods in these organisms served as guide for comparing and identifying orthologous transcription factors in human cells (e.g., TFIIIA, TFIIIB [BDP1], TFIIIC35 [GTF3C6]). However, several of the human RNAP III transcription factors were not cloned by homology, but due to extensive purification from human cells (TFIIIB [BRF2], PTF/SNAPc, TFIIIC [GTF3C1-5]). Purification of these factors by employing biochemical methods was only possible, because functional assays were developed that allowed detecting their specific activities. These assays included in vitro transcription, electrophoretic mobility shift assays (EMSA) and footprinting techniques (descriptions of these techniques are found in 1-3 ). Two techniques were essential for the biochemical purification and functional analysis of human transcription factors: (i) the elaboration of protocols that allowed deriving protein extracts from human cells and (ii) the development of cell-free in vitro transcription assays. [4][5][6] Template DNA for in vitro transcription was provided by genes cloned into plasmid DNA. In the case of RNAP III transcription, usually complete and generally short transcribed sequences were included into the...

Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3′ splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA–mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3′ss. Cryo–electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3′ region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3′ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3′ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3′ss choices.

In Eukaryotes, tRNAs, 5S RNA and U6 RNA are transcribed by RNA polymerase (Pol) III. Human Pol III is composed of 17 subunits. Three specific Pol III subunits form a stable ternary subcomplex (RPC62-RPC39-RPC32α/β) being involved in pre-initiation complex formation. No paralogues for subunits of this subcomplex subunits have been found in Pols I or II, but hRPC62 was shown to be structurally related to the general Pol II transcription factor hTFIIEα. Here we show that these structural homologies extend to functional similarities. hRPC62 as well as hTFIIEα possess intrinsic ATP-dependent 3′-5′ DNA unwinding activity. The ATPase activities of both proteins are stimulated by single-stranded DNA. Moreover, the eWH domain of hTFIIEα can replace the first eWH (eWH1) domain of hRPC62 in ATPase and DNA unwinding assays. Our results identify intrinsic enzymatic activities in hRPC62 and hTFIIEα.