Animal replication-dependent histone pre-mRNAs are processed at the 3= end by endonucleolytic cleavage that is not followed by polyadenylation. The cleavage reaction is catalyzed by CPSF73 and depends on the U7 snRNP and its integral component, Lsm11. A critical role is also played by the 220-kDa protein FLASH, which interacts with Lsm11. Here we demonstrate that the N-terminal regions of these two proteins form a platform that tightly interacts with a unique combination of polyadenylation factors: symplekin, CstF64, and all CPSF subunits, including the endonuclease CPSF73. The interaction is inhibited by alterations in each component of the FLASH/Lsm11 complex, including point mutations in FLASH that are detrimental for processing. The same polyadenylation factors are associated with the endogenous U7 snRNP and are recruited in a U7-dependent manner to histone pre-mRNA. Collectively, our studies identify the molecular mechanism that recruits the CPSF73 endonuclease to histone pre-mRNAs, reveal an unexpected complexity of the U7 snRNP, and suggest that in animal cells polyadenylation factors assemble into two alternative complexes-one specifically crafted to generate polyadenylated mRNAs and the other to generate nonpolyadenylated histone mRNAs that end with the stem-loop.T he vast majority of eukaryotic pre-mRNAs are processed at the 3= end by cleavage coupled to polyadenylation (1-4). In this reaction, pre-mRNAs are cleaved 15 to 30 nucleotides after the highly conserved AAUAAA sequences and the upstream cleavage product is extended by addition of a poly(A) tail. Cleavage coupled to polyadenylation is carried out by a macromolecular machinery consisting of multiple proteins that assemble into at least four separate subcomplexes or factors. The AAUAAA sequence is recognized by cleavage and polyadenylation specificity factor (CPSF), which contains CPSF160, CPSF100, CPSF73, CPSF30, Fip1 (5), and the recently identified WDR33 (6). CPSF160 directly contacts the AAUAAA hexanucleotide, whereas CPSF73 is the endonuclease that catalyzes the cleavage reaction (7). Cleavage stimulation factor (CstF), consisting of CstF77, CstF64, and CstF50, recognizes the GU-rich sequence located downstream of the cleavage site. CstF64 makes direct contacts with this sequence and also interacts with CstF77, which in turn interacts with CstF50 (8). 3=-end processing by cleavage and polyadenylation additionally requires cleavage factor (CF) I m , consisting of 25-kDa and 68-kDa subunits (9), and cleavage factor II m containing at least two subunits, Pcf11 and Clp1 (2, 10). Individual components of the cleavage and polyadenylation machinery are connected with each other through a dense network of protein-protein interactions that stabilizes the entire complex and juxtaposes CPSF73 with the cleavage site. An important role in forming this network is played by symplekin, a protein that interacts with a number of polyadenylation factors and likely functions as a scaffold in 3=-end processing (8,11,12) and other processes, including cytoplasmi...

Maf1 protein is a global negative regulator of RNA polymerase (Pol) III transcription conserved from yeast to man. We report that phosphorylation of Maf1 by casein kinase II (CK2), a highly evolutionarily conserved eukaryotic kinase, is required for efficient Pol III transcription. Both recombinant human and yeast CK2 were able to phosphorylate purified human or yeast Maf1, indicating that Maf1 can be a direct substrate of CK2. Upon transfer of Saccharomyces cerevisiae from repressive to favorable growth conditions, CK2 activity is required for the release of Maf1 from Pol III bound to a tRNA gene and for subsequent activation of tRNA transcription. In a yeast strain lacking Maf1, CK2 inhibition showed no effect on tRNA synthesis, confirming that CK2 activates Pol III via Maf1. Additionally, CK2 was found to associate with tRNA genes, and this association is enhanced in absence of Maf1, especially under repressive conditions. These results corroborate the previously reported TFIIIB-CK2 interaction and indicate an important role of CK2-mediated Maf1 phosphorylation in triggering Pol III activation.RNA polymerase III regulation | transfer RNA | casein kinase II regulation R NA polymerase (Pol) III is responsible for the transcription of some 300 different genes in yeast (class III genes), mostly tRNA genes (1). In-depth analyses of the yeast Pol III transcription system have revealed a cascade of protein-DNA and protein-protein interactions leading to the recruitment of Pol III to its target tRNA genes: binding of the six-subunit TFIIIC factor to the intragenic promoter, TFIIIC-directed recruitment and assembly of the three subunits of TFIIIB (TBP, Brf1, and Bdp1) and subsequent recruitment of the 17-subunit Pol III enzyme (2). High rate of tRNA transcription is achieved through many rounds of reinitiation by Pol III on stable DNA-bound complexes of the initiation factor TFIIIB (3, 4).Pol III is under control of the general negative regulator Maf1 (5, 6), which binds to Pol III clamp and rearranges specific subcomplex C82/34/31, which is required for transcription initiation (7). In the repressive complex, Maf1 impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors TFIIB and thus prevents closed-complex formation (4, 7). Maf1 is essential for repressing Pol III transcription in yeast and mediates several signaling pathways (8). In addition to the down-regulation that occurs normally in the stationary phase, Pol III repression accompanying starvation, respiratory growth, as well as oxidative and replication stress, also requires Maf1 (9-11). Maf1 inhibits Pol III transcription via a mechanism that depends on the dephosphorylation and nuclear accumulation of Maf1 followed by its physical association with Pol III at Pol III-transcribed genes genomewide (6, 12). In contrast Maf1 phosphorylation occurs in favorable growth conditions and is linked to cytoplasmic localization of Maf1 (6, 13).Maf1 was recently found to be phosphorylated by protein kinases PKA (14, 15), Sch9 (16-18), and TO...

Neuronal ceroid lipofuscinoses (NCL) are the most common inherited progressive encephalopathies of childhood. One of the most prevalent forms of NCL, Juvenile neuronal ceroid lipofuscinosis (JNCL) or CLN3 disease (OMIM: 204200), is caused by mutations in the CLN3 gene on chromosome 16p12.1. Despite progress in the NCL field, the primary function of ceroid-lipofuscinosis neuronal protein 3 (CLN3) remains elusive. In this study, we aimed to clarify the role of human CLN3 in the brain by identifying CLN3-associated proteins using a Tandem Affinity Purification coupled to Mass Spectrometry (TAP-MS) strategy combined with Significance Analysis of Interactome (SAINT). Human SH-SY5Y-NTAP-CLN3 stable cells were used to isolate native protein complexes for subsequent TAP-MS. Bioinformatic analyses of isolated complexes yielded 58 CLN3 interacting partners (IP) including 42 novel CLN3 IP, as well as 16 CLN3 high confidence interacting partners (HCIP) previously identified in another high-throughput study by Behrends et al., 2010. Moreover, 31 IP of ceroid-lipofuscinosis neuronal protein 5 (CLN5) were identified (18 of which were in common with the CLN3 bait). Our findings support previously suggested involvement of CLN3 in transmembrane transport, lipid homeostasis and neuronal excitability, as well as link it to G-protein signaling and protein folding/sorting in the ER.

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