Functional Analysis of the Integrator Subunit 12 Identifies a Microdomain That Mediates Activation of the Drosophila Integrator Complex

The Drosophila integrator complex consists of 14 subunits that associate with the C terminus of Rpb1 and catalyze the endonucleolytic cleavage of nascent snRNAs near their 3′ ends. Although disruption of almost any integrator subunit causes snRNA misprocessing, very little is known about the role of the individual subunits or the network of structural and functional interactions that exist within the complex. Here we developed an RNAi rescue assay in Drosophila S2 cells to identify functional domains within integrator subunit 12 (IntS12) required for snRNA 3′ end formation. Surprisingly, the defining feature of the Ints12 protein, a highly conserved and centrally located plant homeodomain finger domain, is not required for reporter snRNA 3′ end cleavage. Rather, we find a small, 45-amino acid N-terminal microdomain to be both necessary and nearly sufficient for snRNA biogenesis in cells depleted of endogenous IntS12 protein. This IntS12 microdomain can function autonomously, restoring full integrator processing activity when introduced into a heterologous protein. Moreover, mutations within the microdomain not only disrupt IntS12 function but also abolish binding to other integrator subunits. Finally, the IntS12 microdomain is sufficient to interact and stabilize the putative scaffold integrator subunit, IntS1. Collectively, these results identify an unexpected interaction between the largest and smallest integrator subunits that is essential for the 3′ end formation of Drosophila snRNA.     

Direct interactions between subunits of CPSF and the U2 snRNP contribute to the coupling of pre-mRNA 3' end processing and splicing

Eukaryotic pre-mRNAs are capped at their 5' ends, polyadenylated at their 3' ends, and spliced before being exported from the nucleus to the cytoplasm. Although the three processing reactions can be studied separately in vitro, they are coupled in vivo. We identified subunits of the U2 snRNP in highly purified CPSF and showed that the two complexes physically interact. We therefore tested whether this interaction contributes to the coupling of 3' end processing and splicing. We found that CPSF is necessary for efficient splicing activity in coupled assays and that mutations in the pre-mRNA binding site of the U2 snRNP resulted in impaired splicing and in much reduced cleavage efficiency. Moreover, we showed that efficient cleavage required the presence of the U2 snRNA in coupled assays. We therefore propose that the interaction between CPSF and the U2 snRNP contributes to the coupling of splicing and 3' end formation.     

A Complex Containing the CPSF73 Endonuclease and Other Polyadenylation Factors Associates with U7 snRNP and Is Recruited to Histone Pre-mRNA for 3′-End Processing

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.     

An RNAi screen identifies additional members of the Drosophila Integrator complex and a requirement for cyclin C/Cdk8 in snRNA 3′-end formation

Formation of the 3′ end of RNA polymerase II–transcribed snRNAs requires a poorly understood group of proteins called the Integrator complex. Here we used a fluorescence-based read-through reporter that expresses GFP in response to snRNA misprocessing and performed a genome-wide RNAi screen in Drosophila S2 cells to identify novel factors required for snRNA 3′-end formation. In addition to the known Integrator complex members, we identified Asunder and CG4785 as additional Integrator subunits. Functional and biochemical experiments revealed that Asunder and CG4785 are additional core members of the Integrator complex. We also identified a conserved requirement in both fly and human snRNA 3′-end processing for cyclin C and Cdk8 that is distinct from their function in the Mediator Cdk8 module. Moreover, we observed biochemical association between Integrator proteins and cyclin C/Cdk8, and that overexpression of a kinase-dead Cdk8 causes snRNA misprocessing. These data functionally define the Drosophila Integrator complex and demonstrate an additional function for cyclin C/Cdk8 unrelated to its function in Mediator.     

The poly A polymerase Star-PAP controls 3'-end cleavage by promoting CPSF interaction and specificity toward the pre-mRNA

Star-PAP is a poly (A) polymerase (PAP) that is putatively required for 3'-end cleavage and polyadenylation of a select set of pre-messenger RNAs (mRNAs), including heme oxygenase (HO-1) mRNA. To investigate the underlying mechanism, the cleavage and polyadenylation of pre-mRNA was reconstituted with nuclear lysates. siRNA knockdown of Star-PAP abolished cleavage of HO-1, and this phenotype could be rescued by recombinant Star-PAP but not PAPα. Star-PAP directly associated with cleavage and polyadenylation specificity factor (CPSF) 160 and 73 subunits and also the targeted pre-mRNA. In vitro and in vivo Star-PAP was required for the stable association of CPSF complex to pre-mRNA and then CPSF 73 specifically cleaved the mRNA at the 3'-cleavage site. This mechanism is distinct from canonical PAPα, which is recruited to the cleavage complex by interacting with CPSF 160. The data support a model where Star-PAP binds to the RNA, recruits the CPSF complex to the 3'-end of pre-mRNA and then defines cleavage by CPSF 73 and subsequent polyadenylation of its target mRNAs.     

Electron microscopy of U4/U6 snRNP reveals a Y-shaped U4 and U6 RNA containing domain protruding from the U4 core RNP

We describe the electron microscopic investigation of purified U4/U6 snRNPs from human and murine cells. The U4/U6 snRNP exhibits two morphological features, a main body approximately 8 nm in diameter and a peripheral filamentous domain, 7-10 nm long. Two lines of evidence suggest that the peripheral domain may consist of RNA and to contain U6 RNA as well as the 5' portion of U4 RNA. (a) Separation of the U4/U6 snRNA interaction regions from the core domains by site-directed cleavage of the U4 snRNA with RNase H gave filament-free, globular core snRNP structures. (b) By immuno and DNA-hybridization EM, both the 5' end of U4 and the 3' end of U6 snRNA were located at the distal region of the filamentous domain, furthest from the core. These results, together with our observation that the filamentous U4/U6 domain is often Y shaped, correlate strikingly with the consensus secondary structure proposed by Brow and Guthrie (1988. Nature (Lond.), 334:213-218), where U4 and U6 snRNA are base paired in such a way that two U4/U6 helices together with a stem/loop of U4 snRNA make up a Y-shaped U4/U6 interaction domain.     

The 5''-terminal sequence of U1 RNA complementary to the consensus 5'' splice site of hnRNA is single-stranded in intact U1 snRNP particles.

The 5'-terminal region of U1 snRNA is highly complementary to the consensus exon-intron regions of hnRNA and it has been suggested that U1 snRNP might play a role in the splicing of the pre-mRNA by intermolecular base-pairing between these regions. Here the secondary structure of the 5' terminus of U1 RNA in the isolated native U1 snRNP particle has been investigated by site-directed enzymatic cleavage of the RNA. Individual oligodeoxynucleotides complementary to various sequences within the first 15 nucleotides of the 5' terminus of U1 RNA have been tested for their ability to form stable DNA X RNA hybrids, with subsequent cleavage of the U1 RNA by RNase H. Our results show unequivocally that the 9 nucleotides at the 5' terminus which are complementary to a consensus 5' splice site are indeed single-stranded in the intact U1 snRNP particle, and are not protected by snRNP proteins. However, they also indicate that the U1 sequence complementary to an intron's consensus 3' end is not readily available for intermolecular base-pairing, either in the intact U1 snRNP particle or in the deproteinized U1 RNA molecule. Therefore our data favour the possibility that U1 snRNP plays a role only in the recognition of a 5' splice site of hnRNA, rather than being involved in the alignment of both ends of an intron for splicing.     

Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus.

Cleavage-polyadenylation specificity factor (CPSF) is one of five separable factors known to be required for 3' cleavage and polyadenylation of mRNA precursors in vitro. Previous studies have shown that the cleavage and poly(A) addition reactions can be uncoupled in vitro and have suggested that CPSF may be the only factor essential for both of these subreactions. Here we report the purification of CPSF to near homogeneity from calf thymus and show that the purified factor contains three polypeptides of 165, 105, and 70 kDa. These polypeptides cosediment precisely with CPSF activity, which has a sedimentation coefficient of 11.5 S. Consistent with previous reports from our laboratory, purified CPSF does not contain a detectable RNA component, indicating that it is a multisubunit protein and not a small nuclear ribonucleoprotein. Extensively purified bovine CPSF can function with human poly(A) polymerase to bring about AAUAAA-dependent poly(A) addition or with human cleavage factors to catalyze accurate 3' cleavage of a pre-mRNA substrate. UV cross-linking and gel retention analyses demonstrate that highly purified CPSF interacts with one of these cleavage factors, the multisubunit cleavage-stimulation factor, to facilitate stable binding of both to an AAUAAA-containing pre-mRNA. Likewise, evidence is presented indicating that poly(A) polymerase and CPSF can interact directly.     

A CPSF-73 homologue is required for cell cycle progression but not cell growth and interacts with a protein having features of CPSF-100

Formation of the mature 3' ends of the vast majority of cellular mRNAs occurs through cleavage and polyadenylation and requires a cleavage and polyadenylation specificity factor (CPSF) containing, among other proteins, CPSF-73 and CPSF-100. These two proteins belong to a superfamily of zinc-dependent beta-lactamase fold proteins with catalytic specificity for a wide range of substrates including nucleic acids. CPSF-73 contains a zinc-binding histidine motif involved in catalysis in other members of the beta-lactamase superfamily, whereas CPSF-100 has substitutions within the histidine motif and thus is unlikely to be catalytically active. Here we describe two previously unknown human proteins, designated RC-68 and RC-74, which are related to CPSF-73 and CPSF-100 and which form a complex in HeLa and mouse cells. RC-68 contains the intact histidine motif, and hence it might be a functional counterpart of CPSF-73, whereas RC-74 lacks this motif, thus resembling CPSF-100. In HeLa cells RC-68 is present in both the cytoplasm and the nucleus whereas RC-74 is exclusively nuclear. RC-74 does not interact with CPSF-73, and neither RC-68 nor RC-74 is found in a complex with CPSF-160, indicating that these two proteins form a separate entity independent of the CPSF complex and are likely involved in a pre-mRNA processing event other than cleavage and polyadenylation of the vast majority of cellular pre-mRNAs. RNA interference-mediated depletion of RC-68 arrests HeLa cells early in G(1) phase, but surprisingly the arrested cells continue growing and reach the size typical of G(2) cells. RC-68 is highly conserved from plants to humans and may function in conjunction with RC-74 in the 3' end processing of a distinct subset of cellular pre-mRNAs encoding proteins required for G(1) progression and entry into S phase.     

Structural analyses of the 7SK ribonucleoprotein (RNP), the most abundant human small RNP of unknown function.

The human 7SK ribonucleoprotein (RNP) has been analyzed to determine its RNA secondary structure and protein constituents. HeLa cell 7SK RNA alone and within its RNP have been probed by chemical modification and enzymatic cleavage, and sites of modification or cleavage have been mapped by primer extension. The resulting secondary structure suggests that structural determinants necessary for capping (a 5' stem followed by the sequence AUPuUPuC) and nuclear migration (the sequence AUPuUPuC) of 7SK RNA may be similar to those for U6 small nuclear RNA (snRNA). It also supports existence of a 3' stem structure which could serve to self-prime cDNA synthesis during pseudogene formation. Oligonucleotide-directed RNase H digestion indicated regions of 7SK RNA capable of base pairing with other nucleic acids. Antisense 2'-O-methyl RNA oligonucleotides were used to affinity select the 7SK RNP from an in vivo 35S-labeled cell sonic extract and identify eight associated proteins of 83, 48, 45, 43, 42, 21, 18, and 13 kDa. 7SK RNA has extensive sequence complementarity to U4 snRNA, within the U4/U6 base pairing domain, and also to U11 snRNA. The possibility that the 7SK RNP is an unrecognized component of the pre-mRNA processing machinery is discussed.     

A multisubunit 3'' end processing factor from yeast containing poly(A) polymerase and homologues of the subunits of mammalian cleavage and polyadenylation specificity factor.

Polyadenylation is the second step in 3' end formation of most eukaryotic mRNAs. In Saccharomyces cerevisiae, this step requires three trans-acting factors: poly(A) polymerase (Pap1p), cleavage factor I (CF I) and polyadenylation factor I (PF I). Here, we describe the purification and subunit composition of a multiprotein complex containing Pap1p and PF I activities. PF I-Pap1p was purified to homogeneity by complementation of extracts mutant in the Fip1p subunit of PF I. In addition to Fip1p and Pap1p, the factor comprises homologues of all four subunits of mammalian cleavage and polyadenylation specificity factor (CPSF), as well as Ptalp, which previously has been implicated in pre-tRNA processing, and several as yet uncharacterized proteins. As expected for a PF I subunit, pta1-1 mutant extracts are deficient for polyadenylation in vitro. PF I also appears to be functionally related to CPSF, as it polyadenylates a substrate RNA more efficiently than Pap1p alone. Possibly, the observed interaction of the complex with RNA tethers Pap1p to its substrate.