This splicing pattern is similar to the results obtained in the SRSF1 expressing cells

er, the order of these subsequent compositional changes remains unclear. A number of studies including very recent single molecule studies in yeast, indicate that the recruitment of the NTC occurs after release of the U4 snRNA, which is consistent with U4/U6 protein release preceeding recruitment of the vast majority of the Prp19/CDC5L complex. Release of the U6 snRNA from U4 allows rearrangements in the structure of the U6 snRNA. The formation of a rearranged U6 structure, including formation of the U6 ISL, may provide new binding sites that enable the recruitment/stable binding of the Prp19/CDC5L complex and related proteins, and/or Bact proteins. In yeast, the NTC was reported to be required for the release of the Lsm proteins from the 30 -end of U6 snRNA, thus the interaction or stable association of the Prp19/CDC5L complex and related proteins with the spliceosome may be a prerequisite for the subsequent loss of the Lsm proteins from the spliceosome during activation. Indeed, recent order Amezinium metilsulfate cryo-EM studies of the S. cerevisiae spliceosomal Bact complex, revealed that several NTC proteins are close to U2/U6 helix II and thus to the 3′ end of the U6 snRNA. As the Lsm proteins bind the 3′ end of U6 Sidarovich et al. eLife 2017;6:e23533. DOI: 10.7554/eLife.23533 14 of 32 Research article Biochemistry Cell Biology , it is likely that their binding and that of the aforementioned NTC proteins is mutually exclusive. A potential role for the Prp19/CDC5 complex and related proteins in establishing the U2/U6 RNA three-way junction in Bact complexes In B028, the U4/U6 duplex has been unwound and U4 snRNA, as well as the U4/U6 snRNP proteins, have been destabilized/released from the spliceosome. This frees U6 snRNA to engage in new intraand inter-molecular base pairing interactions, leading to the catalytically active RNA network within the spliceosome. Our structure probing data suggest that in B028 the U6 snRNA has rearranged and formed the functionally important ISL, but that the lower stem of the U6-ISL is extended by three base pairs. This would prevent formation of U2/U6 helix Ib and is consistent with the formation of a U2/U6 four-way junction. As U2 and U6 have been shown to form a threeway junction in human Bact complexes, the apparently different structure of U2/U6 in B028 may represent an intermediate PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19826959 conformation that forms directly after release of U6 from the U4 duplex by Brr2. This would be consistent with the idea that the catalytic RNA conformation forms stepwise during the B to Bact transition. In yeast, U2 and U6 have been shown to form a three-way junction, but there is also evidence that a competing four-way junction can form, at least in vitro. U2/U6 four-helix junction formation had been documented previously only with protein-free snRNAs, and it is thus likely that spliceosomal proteins promote the formation of a U2/U6 three-helix junction. Subsequent conversion of the apparent U2/U6 four-way junction observed in B028, to a three-way junction, may require the interaction of proteins normally recruited/stabilized during activation, and/or the loss of the Lsm or B-specific proteins, that are normally released prior to Bact formation. In the yeast Bact complex, the catalytic U2/U6 RNA network is contacted not only by the U5 Prp8 protein, but also by Cef1 and Prp46, which are present in the human Prp19/CDC5L complex, and the Prp19/CDC5L-related proteins Syf3, Prp45 and Cwc2 . Given the multiple contacts that the Prp19/CDC5L complex