Little is known about how particle-specific proteins are assembled on spliceosomal small nuclear ribonucleoproteins (snRNPs). Brr2p is a U5 snRNP-specific RNA helicase required for spliceosome catalytic activation and disassembly. In yeast, the Aar2 protein is part of a cytoplasmic precursor U5 snRNP that lacks Brr2p and is replaced by Brr2p in the nucleus. Here we show that Aar2p and Brr2p bind to different domains in the C-terminal region of Prp8p; Aar2p interacts with the RNaseH domain, whereas Brr2p interacts with the Jab1/MPN domain. These domains are connected by a long, flexible linker, but the Aar2p-RNaseH complex sequesters the Jab1/MPN domain, thereby preventing binding by Brr2p. Aar2p is phosphorylated in vivo, and a phospho-mimetic S253E mutation in Aar2p leads to disruption of the Aar2p-Prp8p complex in favor of the Brr2p-Prp8p complex. We propose a model in which Aar2p acts as a phosphorylation-controlled U5 snRNP assembly factor that regulates the incorporation of the particle-specific Brr2p. The purpose of this regulation may be to safeguard against nonspecific RNA binding to Prp8p and/or premature activation of Brr2p activity.[Keywords: pre-mRNA splicing; protein interaction; protein phosphorylation; protein structure; spliceosome; yeast] Supplemental material is available for this article. Small ribonucleoproteins (RNPs) are major components of several RNA processing machineries in eukaryotic cells, including spliceosomes, which catalyze the removal of noncoding intervening sequences (introns) from precursor messenger RNAs (pre-mRNAs) and the ligation of the neighboring coding regions (exons) to generate mature mRNA. Canonical small nuclear RNPs (snRNPs), such as the U1, U2, U4, and U5 snRNPs of the major spliceosome, contain a set of seven common Sm proteins bound at a uridine-rich Sm site in the snRNAs, forming the Sm core RNPs (Pomeranz Krummel et al. 2009;Weber et al. 2010). In metazoans, Sm core RNPs are assembled via an elaborate pathway involving nucleo-cytoplasmic shuttling and two multiprotein machineries, the Prmt5 and the SMN complexes (for review, see Kolb et al. 2007;Chari et al. 2009). In addition, each snRNP contains a variable number of particle-specific proteins. The final stages of metazoan snRNP biogenesis are thought to take place in nuclear Cajal bodies, at least in the case of the U2 snRNP (Nesic et al. 2004). However, little is known about how the specific proteins are assembled.Spliceosomes assemble de novo on the substrate premRNAs by stepwise recruitment of the snRNPs and many additional splicing factors that are not stably associated with snRNPs (for review, see Wahl et al. 2009). During the cycle of assembly, activation, catalysis, and disassembly, the spliceosome is repeatedly remodeled with the help of eight conserved RNA-dependent ATPases/RNA helicases and one G-protein (for review, see Staley and Guthrie 1998). Each remodeling step is associated with changes in the macromolecular composition and in the protein-protein, protein-RNA, and RNA-RNA interaction network...

Yeast U5 small nuclear ribonucleoprotein particle (snRNP) is assembled via a cytoplasmic precursor that contains the U5-specific Prp8 protein but lacks the U5-specific Brr2 helicase. Instead, pre-U5 snRNP includes the Aar2 protein not found in mature U5 snRNP or spliceosomes. Aar2p and Brr2p bind competitively to a C-terminal region of Prp8p that comprises consecutive RNase H-like and Jab1/MPN-like domains. To elucidate the molecular basis for this competition, we determined the crystal structure of Aar2p in complex with the Prp8p RNase H and Jab1/MPN domains. Aar2p binds on one side of the RNase H domain and extends its C terminus to the other side, where the Jab1/MPN domain is docked onto a composite Aar2p-RNase H platform. Known Brr2p interaction sites of the Jab1/ MPN domain remain available, suggesting that Aar2p-mediated compaction of the Prp8p domains sterically interferes with Brr2p binding. Moreover, Aar2p occupies known RNA-binding sites of the RNase H domain, and Aar2p interferes with binding of U4/U6 di-snRNA to the Prp8p C-terminal region. Structural and functional analyses of phosphomimetic mutations reveal how phosphorylation reduces affinity of Aar2p for Prp8p and allows Brr2p and U4/U6 binding. Our results show how Aar2p regulates both protein and RNA binding to Prp8p during U5 snRNP assembly.[Keywords: assembly chaperone; pre-mRNA splicing; regulation by phosphorylation; snRNP biogenesis and recycling; X-ray crystallography] Supplemental material is available for this article. Uridine-rich (U) small nuclear ribonucleoprotein particles (snRNPs) are the main subunits of spliceosomes, the large and dynamic RNP machineries required for the removal of noncoding introns from eukaryotic precursor messenger RNAs (pre-mRNAs) and the ligation of neighboring coding exons. The major spliceosome is built from the U1, U2, U4, U5, and U6 snRNPs, each of which contains an individual small nuclear RNA (snRNA), seven common Sm proteins (or like-Sm [LSm] proteins in the case of U6), and a variable set of particle-specific proteins (for review, see Will and Lü hrmann 2001).A hallmark of the spliceosome is its stepwise assembly from snRNPs and many non-snRNP factors only in the presence of a substrate pre-mRNA (for review, see Wahl et al. 2009). During this process, the spliceosome is repeatedly remodeled with the help of eight highly conserved DEXD/H-box ATPases/RNA helicases (for review, see Staley and Guthrie 1998). Among these enzymes, the U5-specific Brr2 protein is required for the catalytic activation of an initial inactive spliceosomal assembly and again during the ordered disassembly of the postsplicing complex (for review, see Hahn and Beggs 2010). Brr2p is regulated by two other U5 snRNP proteins: Prp8p, considered the master regulator of the spliceosome (for review, see Grainger and Beggs 2005), and Snu114p, a complex G protein that resembles the ribosomal translocase eEF2 (Bartels et al. 2002(Bartels et al. , 2003Brenner and Guthrie 2005;Small et al. 2006).The Sm-type snRNPs themselves are also assemb...

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes.DOI: http://dx.doi.org/10.7554/eLife.06935.001

The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the α-ketoglutarate-dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2–ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2–ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2–ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles.

23The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by 25 the α-ketoglutarate-dependent dioxygenase, AlkBH3. Other ASCC components integrate 26 ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed 27 interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical 28 domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2-ASCC3 29 interfaces are evolutionarily highly conserved and comprise a large number of residues 30 affected by somatic cancer mutations. We quantified contributions of protein regions to the 31 ASCC2-ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2-32 ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation 33 as in the spliceosomal RNA helicase, Brr2. Our results delineate functional regions in an 34 important DNA repair complex and suggest possible molecular disease principles. 35 36 Main 37 The human genome is constantly under assault by endogenous or exogenous DNA 38 damaging agents. To ward off these insults, cells have evolved systems to recognize DNA 39 damage, signal its presence and initiate repair processes. 1 Among the diverse repair 40 mechanisms, direct DNA repair processes represent efficient means to revert chemical 41 changes to DNA and involve enzymes such as photolyases, alkyl-transferases or 42 dioxygenases. 2,3 Escherichia coli α-ketoglutarate-dependent dioxygenase, AlkB, homologs 43 (AlkBH's) are one class of important DNA repair factors that reverse N-alkyl lesions. 4 Among 44 3 the nine identified AlkBH enzymes in human, AlkBH2 and AlkBH3 de-alkylate 1-methyl 45 adenosine and 3-methyl cytosine. 5,6 46Several lines of evidence implicate the human activating signal co-integrator complex 47 (ASCC) in AlkBH3-mediated DNA repair. ASCC is composed of four subunits, ASCC1, ASCC2, 48 ASCC3 and ASC1/TRIP4. 7,8 ASCC3 is the largest subunit of ASCC and was characterized as 49 a DNA helicase that unwinds DNA by translocating on one strand in 3'-to-5' direction. 8 The 50 enzyme is thought to provide single-stranded DNA as a substrate for de-alkylation repair by 51AlkBH3. 8 ASCC and alkylated nucleotides co-localize at nuclear foci upon alkylation damage 52 stress, dependent on a coupling of ubiquitin conjugation to ER degradation (CUE) domain in 53 ASCC2, which links DNA alkylation damage repair to upstream ubiquitin signaling via the RING 54 finger protein 113A. 9 ASCC1 is cleared from these foci upon DNA alkylation damage and 55 knockout of ASCC1 leads to loss of ASCC2 from the nuclear foci and increased cellular 56 sensitivity to alkylating insults. 10 57 Both ASCC2 and ASCC3 have been linked to various human diseases. ASCC2 is 58 upregulated in rheumatoid arthritis patients 11 , and ASCC3 is upregulated in peripheral blood 59 mononuclear cells from lung cancer patients 12,13 . A role of ASCC3 in cancer development or 60 pr...