The transition phase: preparing to launch a laboratory.

The process of starting a laboratory varies between institutions. However, there are universal tasks all investigators will need to address when launching their laboratories. In this piece, we provide a brief summary of considerations for incoming group leaders to centralize this information for the scientific community.

Better late than never: A unique strategy for late gene transcription in the beta- and gammaherpesviruses.

During lytic replication, herpesviruses express their genes in a temporal cascade culminating in expression of "late" genes. Two subfamilies of herpesviruses, the beta- and gammaherpesviruses (including human herpesviruses cytomegalovirus, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus), use a unique strategy to facilitate transcription of late genes. They encode six essential viral transcriptional activators (vTAs) that form a complex at a subset of late gene promoters. One of these vTAs is a viral mimic of host TATA-binding protein (vTBP) that recognizes a strikingly minimal cis-acting element consisting of a modified TATA box with a TATTWAA consensus sequence. vTBP is also responsible for recruitment of cellular RNA polymerase II (Pol II). Despite extensive work in the beta/gammaherpesviruses, the function of the other five vTAs remains largely unknown. The vTA complex and Pol II assemble on the promoter into a viral preinitiation complex (vPIC) to facilitate late gene transcription. Here, we review the properties of the vTAs and the promoters on which they act.

The viral packaging motor potentiates Kaposi's sarcoma-associated herpesvirus gene expression late in infection.

ß- and γ-herpesviruses transcribe their late genes in a manner distinct from host transcription. This process is directed by a complex of viral transcriptional activator proteins that hijack cellular RNA polymerase II and an unknown set of additional factors. We employed proximity labeling coupled with mass spectrometry, followed by CRISPR and siRNA screening to identify proteins functionally associated with the Kaposi's sarcoma-associated herpesvirus (KSHV) late gene transcriptional complex. These data revealed that the catalytic subunit of the viral DNA packaging motor, ORF29, is both dynamically associated with the viral transcriptional activator complex and potentiates gene expression late in infection. Through genetic mutation and deletion of ORF29, we establish that its catalytic activity potentiates viral transcription and is required for robust accumulation of essential late proteins during infection. Thus, we propose an expanded role for ORF29 that encompasses its established function in viral packaging and its newly discovered contributions to viral transcription and late gene expression in KSHV.

DNA processing by the Kaposi's sarcoma-associated herpesvirus alkaline exonuclease SOX contributes to viral gene expression and infectious virion production.

Alkaline exonucleases (AE) are present in several large DNA viruses including bacteriophage λ and herpesviruses, where they play roles in viral DNA processing during genome replication. Given the genetic conservation of AEs across viruses infecting different kingdoms of life, these enzymes likely assume central roles in the lifecycles of viruses where they have yet to be well characterized. Here, we applied a structure-guided functional analysis of the bifunctional AE in the oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV), called SOX. In addition to identifying a preferred DNA substrate preference for SOX, we define key residues important for DNA binding and DNA processing, and how SOX activity on DNA partially overlaps with its functionally separable cleavage of mRNA. By engineering these SOX mutants into KSHV, we reveal roles for its DNase activity in viral gene expression and infectious virion production. Our results provide mechanistic insight into gammaherpesviral AE activity as well as areas of functional conservation between this mammalian virus AE and its distant relative in phage λ.

A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains.

While traditional methods for studying large DNA viruses allow the creation of individual mutants, CRISPR/Cas9 can be used to rapidly create thousands of mutant dsDNA viruses in parallel, enabling the pooled screening of entire viral genomes. Here, we applied this approach to Kaposi's sarcoma-associated herpesvirus (KSHV) by designing a sgRNA library containing all possible ~22,000 guides targeting the 154 kilobase viral genome, corresponding to one cut site approximately every 8 base pairs. We used the library to profile viral sequences involved in transcriptional activation of late genes, whose regulation involves several well characterized features including dependence on viral DNA replication and a known set of viral transcriptional activators. Upon phenotyping all possible Cas9-targeted viruses for transcription of KSHV late genes we recovered these established regulators and identified a new required factor (ORF46), highlighting the utility of the screening pipeline. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we identify the DNA binding but not catalytic domain of ORF46 to be required for viral DNA replication and thus late gene expression. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses.

The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription.

ß- and γ-herpesviruses include the oncogenic human viruses Kaposi's sarcoma-associated virus (KSHV) and Epstein-Barr virus (EBV), and human cytomegalovirus (HCMV), which is a significant cause of congenital disease. Near the end of their replication cycle, these viruses transcribe their late genes in a manner distinct from host transcription. Late gene transcription requires six virally encoded proteins, one of which is a functional mimic of host TATA-box-binding protein (TBP) that is also involved in recruitment of RNA polymerase II (Pol II) via unknown mechanisms. Here, we applied biochemical protein interaction studies together with electron microscopy-based imaging of a reconstituted human preinitiation complex to define the mechanism underlying Pol II recruitment. These data revealed that the herpesviral TBP, encoded by ORF24 in KSHV, makes a direct protein-protein contact with the C-terminal domain of host RNA polymerase II (Pol II), which is a unique feature that functionally distinguishes viral from cellular TBP. The interaction is mediated by the N-terminal domain (NTD) of ORF24 through a conserved motif that is shared in its ß- and γ-herpesvirus homologs. Thus, these herpesviruses employ an unprecedented strategy in eukaryotic transcription, wherein promoter recognition and polymerase recruitment are facilitated by a single transcriptional activator with functionally distinct domains.

Conserved CxnC Motifs in Kaposi's Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34.

Late gene transcription in the beta- and gammaherpesviruses depends on a set of virally encoded transcriptional activators (vTAs) that hijack the host transcriptional machinery and direct it to a subset of viral genes that are required for completion of the viral replication cycle and capsid assembly. In Kaposi's sarcoma-associated herpesvirus (KSHV), these vTAs are encoded by ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. Assembly of the vTAs into a complex is critical for late gene transcription, and thus, deciphering the architecture of the complex is central to understanding its transcriptional regulatory activity. Here, we generated an ORF66-null virus and confirmed that it fails to produce late genes and infectious virions. We show that ORF66 is incorporated into the vTA complex primarily through its interaction with ORF34, which is dependent upon a set of four conserved cysteine-rich motifs in the C-terminal domain of ORF66. While both ORF24 and ORF66 occupy the canonical K8.1 late gene promoter, their promoter occupancy requires the presence of the other vTAs, suggesting that sequence-specific, stable binding requires assembly of the entire complex on the promoter. Additionally, we found that ORF24 expression is impaired in the absence of a stable vTA complex. This work extends our knowledge about the architecture of the KSHV viral preinitiation complex and suggests that it functions as a complex to recognize late gene promoters.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that is the causative agent of multiple human cancers. The release of infectious virions requires the production of capsid proteins and other late genes, whose production is transcriptionally controlled by a complex of six virally encoded proteins that hijack the host transcription machinery. It is poorly understood how this complex assembles or what function five of its six components play in transcription. Here, we demonstrate that ORF66 is an essential component of this complex in KSHV and that its inclusion in the complex depends upon its C-terminal domain, which contains highly conserved cysteine-rich motifs reminiscent of zinc finger motifs. Additionally, we examined the assembly of the viral preinitiation complex at late gene promoters and found that while sequence-specific binding of late gene promoters requires ORF24, it additionally requires a fully assembled viral preinitiation complex.

The life of U6 small nuclear RNA, from cradle to grave.

Removal of introns from precursor messenger RNA (pre-mRNA) and some noncoding transcripts is an essential step in eukaryotic gene expression. In the nucleus, this process of RNA splicing is carried out by the spliceosome, a multi-megaDalton macromolecular machine whose core components are conserved from yeast to humans. In addition to many proteins, the spliceosome contains five uridine-rich small nuclear RNAs (snRNAs) that undergo an elaborate series of conformational changes to correctly recognize the splice sites and catalyze intron removal. Decades of biochemical and genetic data, along with recent cryo-EM structures, unequivocally demonstrate that U6 snRNA forms much of the catalytic core of the spliceosome and is highly dynamic, interacting with three snRNAs, the pre-mRNA substrate, and >25 protein partners throughout the splicing cycle. This review summarizes the current state of knowledge on how U6 snRNA is synthesized, modified, incorporated into snRNPs and spliceosomes, recycled, and degraded.

Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly.

Base-pairing of U4 and U6 snRNAs during di-snRNP assembly requires large-scale remodeling of RNA structure that is chaperoned by the U6 snRNP protein Prp24. We investigated the mechanism of U4/U6 annealing in vitro using an assay that enables visualization of ribonucleoprotein complexes and faithfully recapitulates known in vivo determinants for the process. We find that annealing, but not U6 RNA binding, is highly dependent on the electropositive character of a 20 Å-wide groove on the surface of Prp24. During annealing, we observe the formation of a stable ternary complex between U4 and U6 RNAs and Prp24, indicating that displacement of Prp24 in vivo requires additional factors. Mutations that stabilize the U6 'telestem' helix increase annealing rates by up to 15-fold, suggesting that telestem formation is rate-limiting for U4/U6 pairing. The Lsm2-8 complex, which binds adjacent to the telestem at the 3' end of U6, provides a comparable rate enhancement. Collectively, these data identify domains of the U6 snRNP that are critical for one of the first steps in assembly of the megaDalton U4/U6.U5 tri-snRNP complex, and lead to a dynamic model for U4/U6 pairing that involves a striking degree of evolved cooperativity between protein and RNA.

A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex.

The small nuclear RNA (snRNA) components of the spliceosome undergo many conformational rearrangements during its assembly, catalytic activation and disassembly. The U4 and U6 snRNAs are incorporated into the spliceosome as a base-paired complex within the U4/U6.U5 small nuclear ribonucleoprotein (tri-snRNP). U4 and U6 are then unwound in order for U6 to pair with U2 to form the spliceosome's active site. After splicing, U2/U6 is unwound and U6 annealed to U4 to reassemble the tri-snRNP. U6 rearrangements are crucial for spliceosome formation but are poorly understood. We have used single-molecule Förster resonance energy transfer and unwinding assays to identify interactions that promote U4/U6 unwinding and have studied their impact in yeast. We find that U4/U6 is efficiently unwound using DNA oligonucleotides by coupling unwinding of U4/U6 stem II with strand invasion of stem I. Unwinding is stimulated by the U6 telestem, which transiently forms in the intact U4/U6 RNA complex. Stabilization of the telestem in vivo results in accumulation of U4/U6 di-snRNP and impairs yeast growth. Our data reveal conserved mechanisms for U4/U6 unwinding and indicate telestem dynamics are critical for tri-snRNP assembly and stability.

Structure of Pisum sativum Rubisco with bound ribulose 1,5-bisphosphate.

The first structure of a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from a pulse crop is reported. Rubisco was purified from Pisum sativum (garden pea) and diffraction-quality crystals were obtained by hanging-drop vapour diffusion in the presence of the substrate ribulose 1,5-bisphosphate. X-ray diffraction data were recorded to 2.20 Šresolution from a single crystal at the Canadian Light Source. The overall quaternary structure of non-activated P. sativum Rubisco highlights the conservation of the form I Rubisco hexadecameric complex. The electron density places the substrate in the active site at the interface of the large-subunit dimers. Lys201 in the active site is not carbamylated as expected for this non-activated structure. Some heterogeneity in the small-subunit sequence is noted, as well as possible variations in the conformation and contacts of ribulose 1,5-bisphosphate in the large-subunit active sites. Overall, the active-site conformation most closely correlates with the `closed' conformation observed in other substrate/inhibitor-bound Rubisco structures.

A pentameric protein ring with novel architecture is required for herpesviral packaging.

Genome packaging in large double-stranded DNA viruses requires a powerful molecular motor to force the viral genome into nascent capsids, which involves essential accessory factors that are poorly understood. Here, we present structures of two such accessory factors from the oncogenic herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV; ORF68) and Epstein-Barr virus (EBV; BFLF1). These homologous proteins form highly similar homopentameric rings with a positively charged central channel that binds double-stranded DNA. Mutation of individual positively charged residues within but not outside the channel ablates DNA binding, and in the context of KSHV infection, these mutants fail to package the viral genome or produce progeny virions. Thus, we propose a model in which ORF68 facilitates the transfer of newly replicated viral genomes to the packaging motor.

Architecture of the U6 snRNP reveals specific recognition of 3'-end processed U6 snRNA.

The spliceosome removes introns from precursor messenger RNA (pre-mRNA) to produce mature mRNA. Prior to catalysis, spliceosomes are assembled de novo onto pre-mRNA substrates. During this assembly process, U6 small nuclear RNA (snRNA) undergoes extensive structural remodeling. The early stages of this remodeling process are chaperoned by U6 snRNP proteins Prp24 and the Lsm2-8 heteroheptameric ring. We now report a structure of the U6 snRNP from Saccharomyces cerevisiae. The structure reveals protein-protein contacts that position Lsm2-8 in close proximity to the chaperone "active site" of Prp24. The structure also shows how the Lsm2-8 ring specifically recognizes U6 snRNA that has been post-transcriptionally modified at its 3' end, thereby elucidating the mechanism by which U6 snRNPs selectively recruit 3' end-processed U6 snRNA into spliceosomes. Additionally, the structure reveals unanticipated homology between the C-terminal regions of Lsm8 and the cytoplasmic Lsm1 protein involved in mRNA decay.

Structure and conformational plasticity of the U6 small nuclear ribonucleoprotein core.

U6 small nuclear RNA (snRNA) is a key component of the active site of the spliceosome, a large ribonucleoprotein complex that catalyzes the splicing of precursor messenger RNA. Prior to its incorporation into the spliceosome, U6 is bound by the protein Prp24, which facilitates unwinding of the U6 internal stem-loop (ISL) so that it can pair with U4 snRNA. A previously reported crystal structure of the `core' of the U6 small nuclear ribonucleoprotein (snRNP) contained an ISL-stabilized A62G mutant of U6 bound to all four RNA-recognition motif (RRM) domains of Prp24 [Montemayor et al. (2014), Nature Struct. Mol. Biol. 21, 544-551]. The structure revealed a novel topology containing interlocked rings of protein and RNA that was not predicted by prior biochemical and genetic data. Here, the crystal structure of the U6 snRNP core with a wild-type ISL is reported. This complex crystallized in a new space group, apparently owing in part to the presence of an intramolecular cross-link in RRM1 that was not observed in the previously reported U6-A62G structure. The structure exhibits the same protein-RNA interface and maintains the unique interlocked topology. However, the orientation of the wild-type ISL is altered relative to the A62G mutant structure, suggesting inherent structural dynamics that may facilitate its pairing with U4. Consistent with their similar architectures in the crystalline state, the wild-type and A62G variants of U6 exhibit similar Prp24-binding affinities and electrophoretic mobilities when analyzed by gel-shift assay.

Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities.

U6 small nuclear ribonucleoprotein (snRNP) biogenesis is essential for spliceosome assembly, but not well understood. Here, we report structures of the U6 RNA processing enzyme Usb1 from yeast and a substrate analog bound complex from humans. Unlike the human ortholog, we show that yeast Usb1 has cyclic phosphodiesterase activity that leaves a terminal 3' phosphate which prevents overprocessing. Usb1 processing of U6 RNA dramatically alters its affinity for cognate RNA-binding proteins. We reconstitute the post-transcriptional assembly of yeast U6 snRNP in vitro, which occurs through a complex series of handoffs involving 10 proteins (Lhp1, Prp24, Usb1 and Lsm2-8) and anti-cooperative interactions between Prp24 and Lhp1. We propose a model for U6 snRNP assembly that explains how evolutionarily divergent and seemingly antagonistic proteins cooperate to protect and chaperone the nascent snRNA during its journey to the spliceosome.The mechanism of U6 small nuclear ribonucleoprotein (snRNP) biogenesis is not well understood. Here the authors characterize the enzymatic activities and structures of yeast and human U6 RNA processing enzyme Usb1, reconstitute post-transcriptional assembly of yeast U6 snRNP in vitro, and propose a model for U6 snRNP assembly.

Structural Analysis of Multi-Helical RNAs by NMR-SAXS/WAXS: Application to the U4/U6 di-snRNA.

NMR and SAXS (small-angle X-ray scattering)/WAXS (wide-angle X-ray scattering) are highly complementary approaches for the analysis of RNA structure in solution. Here we describe an efficient NMR-SAXS/WAXS approach for structural investigation of multi-helical RNAs. We illustrate this approach by determining the overall fold of a 92-nt 3-helix junction from the U4/U6 di-snRNA. The U4/U6 di-snRNA is conserved in eukaryotes and is part of the U4/U6.U5 tri-snRNP, a large ribonucleoprotein complex that comprises a major subunit of the assembled spliceosome. Helical orientations can be determined by X-ray scattering data alone, but the addition of NMR RDC (residual dipolar coupling) restraints improves the structure models. RDCs were measured in two different external alignment media and also by magnetic susceptibility anisotropy. The resulting alignment tensors are collinear, which is a previously noted problem for nucleic acids. Including WAXS data in the calculations produces models with significantly better fits to the scattering data. In solution, the U4/U6 di-snRNA forms a 3-helix junction with a planar Y-shaped structure and has no detectable tertiary interactions. Single-molecule Förster resonance energy transfer data support the observed topology. A comparison with the recently determined cryo-electron microscopy structure of the U4/U6.U5 tri-snRNP illustrates how proteins scaffold the RNA and dramatically alter the geometry of the U4/U6 3-helix junction.