Primary triple negative breast cancers (TNBC) represent approximately 16% of all breast cancers1 and are a tumour type defined by exclusion, for which comprehensive landscapes of somatic mutation have not been determined. Here we show in 104 early TNBC cases, that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some exhibiting only a handful of somatic aberrations in a few pathways, whereas others contain hundreds of somatic events and multiple pathways implicated. Integration with matched whole transcriptome sequence data revealed that only ~36% of mutations are expressed. By examining single nucleotide variant (SNV) allelic abundance derived from deep re-sequencing (median >20,000 fold) measurements in 2414 somatic mutations, we determine for the first time in an epithelial tumour, the relative abundance of clonal genotypes among cases in the population. We show that TNBC vary widely and continuously in their clonal frequencies at the time of diagnosis, with basal subtype TNBC2,3 exhibiting more variation than non-basal TNBC. Although p53 and PIK3CA/PTEN somatic mutations appear clonally dominant compared with other pathways, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal and cell shape/motility proteins occurred at lower clonal frequencies, suggesting they occurred later during tumour progression. Taken together our results show that future attempts to dissect the biology and therapeutic responses of TNBC will require the determination of individual tumour clonal genotypes.

Downloaded from 5p11ce050me a55em61y 1n yea5t 5nRNA5 have 6een 5h0wn t0 6e a550c1ated w1th the 5p11-ce050me (P1k1e1ny et a1. 1986., 2. H05t0m5ky et a1., unpu61.; N. R1ede1 et a1., per5. c0mm.}, and we 5ha11 pr0-v1de further ev1dence f0r 5uch a550c1at10n5 1n th15 paper. 7he c011ect10n 0f yea5t 5nRNA5 115ted 1n 7a61e 1 have 6een further 1mp11cated 1n mRNA 5p11c1n9 6ecau5e they are prec1p1tated 6y a human aut01mmune ant15erum 5m 1M. J0ne5 and C. 6uthr1e, per5. c0mm.} a5 are the1r mamma11an c0unterpart5. 7h15 ant15erum rec09n12e5 a pr0te1n c0mp0nent wh1ch 15 c0mm0n t0 a11 f1ve U5nRNP5 1nv01ved 1n 5p11c1n9. 7he pr0te1n5 re4u1red f0r mRNA 5p11c1n9 have 6een 1e55 5tud1ed, 6ut 1n yea5t a 5et 0f we11-5tud1ed 9ene5, the RNA 9ene5, enC0de pr0te1n c0mp0nent5 0f the mRNA 5p11c1n9 apparatu5 (Lu5t19 et a1. 1986}. A yea5t t5 5tra1n mutant 1n 0ne 0f the5e 9ene5, ma2, ha5 pr0v1ded 50me 1nf0rmat10n a60ut the a55em61y 0f the 5p11ce050me.U51n9 the rna2 mutant extract, we have 5h0wn that the 5p11ce050me 15 a true 1ntermed1ate 1n the 5p11c1n9 pathway (L1n et a1. 1987}. 7he5e exper1ment5 a150 dem0n5trate that extr1n51c pr0te1n5 act 0n the fu11y a55em-61ed 5p11ce050me. We w111 further 5h0w 1n th15 paper that heat-1nact1vated rna2 mutant extract5 accumu1ate an 1dent1f1a61e a55em61y 1ntermed1ate. 5p11c1n9 c0mp1exe5 can 6e re501ved 6y e1ectr0ph0re515 1n n0ndenatur1n9 p01yacry1am1de 9e15 (K0nar5ka and 5harp 1986; P1k1e1ny et a1. 1986; K0nar5ka and 5harp 1987). 1n th15 paper we u5e that techn14ue t0 1dent1fy f0ur 5p11c1n9-5pec1f1c c0mp1exe5 1n the a55em61y 0f the yea5t 5p11ce050me. U51n9 the 5ame 9e1 5y5tem, we have d15c0vered that three 0f the 5nRNA5 f0und 1n the5e c0mp1exe5, 5nR6, 5nR7, and 5nR14, a550c1ate 1n extract5 t0 f0rm a 1ar9e c0mp1ex. 7h15 c0mp1ex under90e5 an A7P-dependent d1550c1at10n. Re5u1t51dent1f1cat10n 0f tw0 5p11c1n9 c0mp1exe5, A and 8 70 5tudy the f0rmat10n 0f the 5p11ce050me, we have fract10nated 5p11c1n9 react10n m1xture5 0n n0ndena-tur1n9 p01yacry1am1de 9e15. 7he 5u65trate5 1n the5e exper1ment5 were a2P-1a6e1ed 5P6 RNA p01ymera5e tran5cr1pt5 0f a p0rt10n 0f the yea5t act1n 9ene. 5u65trate5 c0nta1n1n9 mutat10n5 1n the act1n 1ntr0n were a150 u5ed. 7he5e mutat10n5 were c0n5tructed 1n a 6ack9r0und 1n wh1ch a crypt1c 7AC7AAC 5e4uence had 6een de1eted 1A6 act1n) (V1jayra9havan et a1. 19861. Hepar1n wa5 added t0 term1nate the 5p11c1n9 react10n and t0 e11m1nate n0n-5pec1f1c 61nd1n9 0f pr0te1n5 t0 the RNA5 (K0nar5ka and 5harp 1986}.1n1t1a11y, tw0 c0mp1exe5 were 065erved dur1n9 the c0ur5e 0f the react10n {F19. 1A). C0mp1ex 8 appeared after 1 m1n 0f 1ncu6at10n f0110wed 6y c0mp1ex A. 80th 0f the5e c0mp1exe5 are 5pec1f1c f0r 5p11c1n9 6ecau5e ne1ther f0rmed w1th an ant15en5e act1n tran5cr1pt 0r w1th RNA c0nta1n1n9 an a1tered 5• 5p11ce 51te (A1 0r C1} 0r 7AC-7AAC 5e4uence (A256, A257, 0r C259). W1th the 3• 5p11ce 51te mutat10n {ACAC}, the f1r5t 5tep 0f 5p11c1n9 can 0ccur 6ut the 5ec0nd 5tep 15 610cked. 7h15 5u65trate can f0rm 5p11ce050me5 a5 detected 6y 91ycer01 9rad1ent 5ed1mentat10n {V1jayra9havan et a1. 1986}. When th15 5u65tr...

Next-generation sequencing of follicular lymphoma and diffuse-large B-cell lymphoma has revealed frequent somatic, heterozygous Y641 mutations in the histone methyltransferase EZH2. Heterozygosity and the presence of equal quantities of both mutant and wild-type mRNA and expressed protein suggest a dominant mode of action. Surprisingly, B-cell lymphoma cell lines and lymphoma samples harboring heterozygous EZH2 Y641 mutations have increased levels of histone H3 Lys-27-specific trimethylation (H3K27me3). Expression of EZH2 Y641F/N mutants in cells with EZH2 WT resulted in an increase of H3K27me3 levels in vivo. Structural modeling of EZH2 Y641 mutants suggests a "Tyr/Phe switch" model whereby structurally neutral, nontyrosine residues at position 641 would decrease affinity for unmethylated and monomethylated H3K27 substrates and potentially favor trimethylation. We demonstrate, using in vitro enzyme assays of reconstituted PRC2 complexes, that Y641 mutations result in a decrease in monomethylation and an increase in trimethylation activity of the enzyme relative to the wild-type enzyme. This represents the first example of a diseaseassociated gain-of-function mutation in a histone methyltransferase, whereby somatic EZH2 Y641 mutations in lymphoma act dominantly to increase, rather than decrease, histone methylation. The dominant mode of action suggests that allelespecific EZH2 inhibitors should be a future therapeutic strategy for this disease. IntroductionNon-Hodgkin lymphomas represent a diverse spectrum of distinct entities with the 2 most common types represented by follicular lymphoma and diffuse large B-cell lymphoma (DLBCL). There are 2 molecular subtypes of DLBCL based on cell-of-origin distinctions: the activated B-cell type and the germinal center B-cell (GCB) type. Both follicular lymphoma and the GCB subtype of DLBCL derive from germinal center B cells. We have shown that, in 7% of follicular lymphomas and 22% of GCB-type DLBCL, a single point mutation in EZH2, which results in a single amino-acid change at position 641, is selected for; EZH2 (Tyr641 or WT) was mutated to phenylalanine (Y641F), asparagine (Y641N), histidine (Y641H), or serine (Y641S). 1 EZH2 has been implicated as an oncoprotein often overexpressed in many solid tumors. [2][3][4] Initial analysis of the activity of Y641 variants in cell-free reconstituted Polycomb Repressive Complex 2 (PRC2) complexes using unmethylated peptides suggested that the mutations behaved as a loss of function. 1 EZH2 is the catalytic member of the PRC2; however, EZH2 alone has very weak histone-methylating activity. Other members of the PRC2 complex include EED, SUZ12, AEBP2, and RbAp48 and are required for full activity. The PRC2 complex has been shown to exhibit in vitro enzyme activity on histone peptide substrates and nucleosomes. EZH2 is a member of the Su(var)3,9, enhancer of zest, Trithorax (SET) domain containing family of histone methyltransferases (HMTases); all contain a conserved SET domain. Genetic and biochemical analysis of EZH2 SET domain ...

During spliceosome activation, a large structural rearrangement occurs that involves the release of two small nuclear RNAs, U1 and U4, and the addition of a protein complex associated with Prp19p. We show here that the Prp19p-associated complex is required for stable association of U5 and U6 with the spliceosome after U4 is dissociated. Ultraviolet crosslinking analysis revealed the existence of two modes of base pairing between U6 and the 5' splice site, as well as a switch of such base pairing from one to the other that required the Prp19p-associated complex during spliceosome activation. Moreover, a Prp19p-dependent structural change in U6 small nuclear ribonucleoprotein particles was detected that involves destabilization of Sm-like (Lsm) proteins to bring about interactions between the Lsm binding site of U6 and the intron sequence near the 5' splice site, indicating dynamic association of Lsm with U6 and a direct role of Lsm proteins in activation of the spliceosome.

The assembly of the spliceosome involves dynamic rearrangements of interactions between snRNAs, protein components, and the pre-mRNA substrate. DExD/H-box ATPases are required to mediate structural changes of the spliceosome, utilizing the energy of ATP hydrolysis. Two DExD/H-box ATPases are required for the catalytic steps of the splicing pathway, Prp2 for the first step and Prp16 for the second step, both belonging to the DEAH subgroup of the protein family. The detailed mechanism of their action was not well understood until recently, when Prp2 was shown to be required for the release of U2 components SF3a and SF3b, presumably to allow the binding of Cwc25 to promote the first transesterification reaction. We show here that Cwc25 and Yju2 are released after the reaction in Prp16-and ATP-dependent manners, possibly to allow for the binding of Prp22, Prp18, and Slu7 to promote the second catalytic reaction. The binding of Cwc25 to the spliceosome is destabilized by mutations at the branchpoint sequence, suggesting that Cwc25 may bind to the branch site. We also show that Prp16 has an ATP-independent role in the first catalytic step, in addition to its known role in the second step. In the absence of ATP, Prp16 stabilizes the binding of Cwc25 to the spliceosome formed with branchpoint mutated pre-mRNAs to facilitate their splicing. Our results uncovered novel functions of Prp16 in both catalytic steps, and provide mechanistic insights into splicing catalysis.