Inefficient Codon Usage Impairs mRNA Accumulation: the Case of the v-FLIP Gene of Kaposi's Sarcoma-Associated Herpesvirus.

UNLABELLED: Latent Kaposi's sarcoma-associated herpesvirus (KSHV) genomes encode a homolog of cellular FLICE-inhibitory proteins (termed v-FLIP) that activates NF-κB and can trigger important proinflammatory and antiapoptotic changes in latently infected cells. The protein is present at very low levels in infection and has generally been difficult to efficiently express in recombinant vectors. Here we show that codon usage in the v-FLIP gene is strikingly suboptimal. Optimization of codon use in expression vectors, as expected, restores efficient protein expression. Surprisingly, however, it also dramatically increases the steady-state level of v-FLIP mRNA, at least in part by increasing mRNA stability. When codon-optimized v-FLIP sequences are reintroduced into intact KSHV genomes, the resulting virus expresses readily detectable monocistronic v-FLIP mRNAs that are undetectable in wild-type (WT) infection by blot hybridization, suggesting that such RNAs are in fact transcribed in WT infection but fail to accumulate. The overexpression of v-FLIP by codon-optimized latent genomes results in a 5- to 7-fold decrement in virus production following lytic induction, indicating that maximizing NF-κB signaling is deleterious to induction. These studies provide a clear explanation for the evolution of inefficient codon usage in this gene and point to a strong connection between translational efficiency and RNA accumulation in mammalian cells. IMPORTANCE: This study reports that inefficient codon usage in a herpesviral gene is strikingly correlated with the inability of its mRNA to accumulate in cells; correction of efficient translatability restores RNA abundance. A similar correlation has been reported in yeast species, but the mechanisms operating in mammalian cells appear substantially different.

KSHV 2.0: a comprehensive annotation of the Kaposi's sarcoma-associated herpesvirus genome using next-generation sequencing reveals novel genomic and functional features.

Productive herpesvirus infection requires a profound, time-controlled remodeling of the viral transcriptome and proteome. To gain insights into the genomic architecture and gene expression control in Kaposi's sarcoma-associated herpesvirus (KSHV), we performed a systematic genome-wide survey of viral transcriptional and translational activity throughout the lytic cycle. Using mRNA-sequencing and ribosome profiling, we found that transcripts encoding lytic genes are promptly bound by ribosomes upon lytic reactivation, suggesting their regulation is mainly transcriptional. Our approach also uncovered new genomic features such as ribosome occupancy of viral non-coding RNAs, numerous upstream and small open reading frames (ORFs), and unusual strategies to expand the virus coding repertoire that include alternative splicing, dynamic viral mRNA editing, and the use of alternative translation initiation codons. Furthermore, we provide a refined and expanded annotation of transcription start sites, polyadenylation sites, splice junctions, and initiation/termination codons of known and new viral features in the KSHV genomic space which we have termed KSHV 2.0. Our results represent a comprehensive genome-scale image of gene regulation during lytic KSHV infection that substantially expands our understanding of the genomic architecture and coding capacity of the virus.

PRICE: software for the targeted assembly of components of (Meta) genomic sequence data.

Low-cost DNA sequencing technologies have expanded the role for direct nucleic acid sequencing in the analysis of genomes, transcriptomes, and the metagenomes of whole ecosystems. Human and machine comprehension of such large datasets can be simplified via synthesis of sequence fragments into long, contiguous blocks of sequence (contigs), but most of the progress in the field of assembly has focused on genomes in isolation rather than metagenomes. Here, we present software for paired-read iterative contig extension (PRICE), a strategy for focused assembly of particular nucleic acid species using complex metagenomic data as input. We describe the assembly strategy implemented by PRICE and provide examples of its application to the sequence of particular genes, transcripts, and virus genomes from complex multicomponent datasets, including an assembly of the BCBL-1 strain of Kaposi's sarcoma-associated herpesvirus. PRICE is open-source and available for free download (derisilab.ucsf.edu/software/price/ or sourceforge.net/projects/pricedenovo/).

Regulation of KSHV lytic switch protein expression by a virus-encoded microRNA: an evolutionary adaptation that fine-tunes lytic reactivation.

Herpesviruses encode numerous microRNAs (miRNAs), most of whose functions are unknown. The Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 17 known miRNAs as part of its latency program, suggesting that these RNAs might function to regulate the latent state. Here we show that one of these KSHV miRNAs, miRK9( *), targets a sequence in the 3' untranslated region (UTR) of the mRNA encoding the major lytic switch protein (RTA), which controls viral reactivation from latency. Ectopic expression of miRK9( *) impairs RTA synthesis, while its specific antagonism in latently infected cells enhances spontaneous lytic reactivation frequency by 2- to 3-fold. Mutation of the recognition sequence in the RTA 3'UTR abolishes RTA downregulation by miRK9( *). We propose that miRNA targeting of RTA, while not the primary regulator of the lytic switch, functions like a safety mechanism on the trigger of lytic reactivation, preventing stochastic variations in basal RTA transcription from activating inappropriate entry into the lytic cycle.

Silencing by small RNAs is linked to endosomal trafficking.

Small RNAs direct RNA-induced silencing complexes (RISCs) to regulate stability and translation of mRNAs. RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies. However, RISC proteins can associate with membrane compartments such as the Golgi and endoplasmic reticulum. Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs). Blocking the maturation of MVBs into lysosomes by loss of the tethering factor HPS4 (ref. 5) enhances short interfering RNA (siRNA)- and micro RNA (miRNA)-mediated silencing in Drosophila melanogaster and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT (endosomal sorting complex required for transport) depletion results in impaired miRNA silencing and loss of GW-bodies. These results indicate that active RISCs are physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISCs in loading small RNAs. We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm.

A role for ubiquitin in the spliceosome assembly pathway.

The spliceosome uses numerous strategies to regulate its function in mRNA maturation. Ubiquitin regulates many cellular processes, but its potential roles during splicing are unknown. We have developed a new strategy that reveals a direct role for ubiquitin in the dynamics of splicing complexes. A ubiquitin mutant (I44A) that can enter the conjugation pathway but is compromised in downstream functions diminishes splicing activity by reducing the levels of the U4/U6-U5 small nuclear ribonucleoprotein (snRNP). Similarly, an inhibitor of ubiquitin's protein-protein interactions, ubistatin A, reduces U4/U6-U5 triple snRNP levels in vitro. When ubiquitin interactions are blocked, ATP-dependent disassembly of purified U4/U6-U5 particles is accelerated, indicating a direct role for ubiquitin in repressing U4/U6 unwinding. Finally, we show that the conserved splicing factor Prp8 is ubiquitinated within purified triple snRNPs. These results reveal a previously unknown ubiquitin-dependent mechanism for controlling the pre-mRNA splicing pathway.

Ubiquitin binding by a variant Jab1/MPN domain in the essential pre-mRNA splicing factor Prp8p.

The U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) are components of the spliceosome, which catalyzes pre-mRNA splicing. One of the largest and the most highly conserved proteins in the spliceosome is Prp8p, a component of the U5 snRNP. Despite its size and conservation, very few motifs have been identified that suggest specific biochemical functions. A variant of the Jab1/MPN domain found in a class of deubiquitinating enzymes is present near the C terminus of Prp8p. Ubiquitination regulates a broad range of cellular pathways, and its functions generally require ubiquitin recognition by one or more ubiquitin-binding domains (UBDs). No precise role for ubiquitin has been defined in the pre-mRNA splicing pathway, and no known UBDs have been found within splicing proteins. Here we show that a Prp8p fragment containing the Jab1/MPN domain binds directly to ubiquitin with an affinity comparable to other known UBDs. Several mutations within this domain that compromise splicing also reduce interaction of the fragment with ubiquitin-Sepharose. Our results define a new UBD and suggest functional links between ubiquitin and the pre-mRNA splicing machinery.