Differential effects of nucleotide analogs on scanning-dependent initiation and elongation of mammalian mRNA translation in vitro

Codon–anticodon interactions are central to both the initiation and elongation phases of eukaryotic mRNA translation. The obvious difference is that the interaction takes place in the ribosomal A-site during elongation, whereas the 40S ribosomal subunit and associated initiation factors scan the mRNA sequence in search of an initiation codon with Met-tRNAi bound in the P-site, ceasing once codon–anticodon interaction is established at the AUG. As an indirect test of whether the two mechanisms of mRNA sequence inspection are basically similar or not, the effects of six different uridine analog substitutions in the mRNA were examined in reticulocyte lysate translation assays and 80S initiation complex formation assays. Four constructs, each with the same reporter coding sequence, were used, differing in whether the initiation codon was AUG or ACG, and in whether the 5′-UTR had U residues or not. Three analogs (5-bromoU, 5-aminoallylU, and pseudoU) inhibited both elongation and initiation, but the other three had striking differential effects. Ribothymidine had a negligible effect on elongation but caused a ∼50% inhibition of initiation, with little effect on actual AUG recognition, which implies that inhibition must have occurred at some earlier step in initiation. In complete contrast, 2′ deoxyU was prohibitive to elongation but had no effect on initiation, and 4-thioU actually stimulated initiation but quite strongly inhibited elongation processivity. These results show that the detailed mechanisms of inspection of the mRNA sequence during scanning-dependent initiation and elongation must be considerably different

Reenacting the birth of an intron

An intron is an extended genomic feature whose function requires multiple constrained positions - donor and acceptor splice sites, a branch point, a polypyrimidine tract and suitable splicing enhancers - that may be distributed over hundreds or thousands of nucleotides. New introns are therefore unlikely to emerge by incremental accumulation of functional sub-elements. Here we demonstrate that a functional intron can be created de novo in a single step by a segmental genomic duplication. This experiment recapitulates in vivo the birth of an intron that arose in the ancestral jawed vertebrate lineage nearly half a billion years ago

Identification and experimental validation of splicing regulatory elements in Drosophila melanogaster reveals functionally conserved splicing enhancers in metazoans

It has been established that alternative and constitutive splicing decisions are dictated in part by cis-acting RNA sequences known as splicing enhancers or inhibitors. Both types of elements can be found either in exons or introns. Here, the authors have used computational methods to identify predicted intronic and exonic splicing enhancers in Drosophila. Many of these were previously shown to be enhancers of vertebrates. The evolutionary implications of these findings are discussed

Kaposi’s sarcoma-associated herpesvirus induces specialised ribosomes to efficiently translate viral lytic mRNAs

Viruses rely on the translational machinery of the host cell to synthesis viral proteins. We show that KSHV manipulates the composition of host cell ribosomes creating a specialised ribosome to specifically translate viral mRNAs

The Drosophila Splicing Factor PSI Is Phosphorylated by Casein Kinase II and Tousled-Like Kinase

<div><p>Alternative splicing of pre-mRNA is a highly regulated process that allows cells to change their genetic informational output. These changes are mediated by protein factors that directly bind specific pre-mRNA sequences. Although much is known about how these splicing factors regulate pre-mRNA splicing events, comparatively little is known about the regulation of the splicing factors themselves. Here, we show that the <i>Drosophila</i> splicing factor P element Somatic Inhibitor (PSI) is phosphorylated at at least two different sites by at minimum two different kinases, casein kinase II (CK II) and tousled-like kinase (tlk). These phosphorylation events may be important for regulating protein-protein interactions involving PSI. Additionally, we show that PSI interacts with several proteins in <i>Drosophila</i> S2 tissue culture cells, the majority of which are splicing factors.</p> </div

m⁶A Regulates the Stability of Cellular Transcripts Required for Efficient KSHV Lytic Replication

The epitranscriptomic modification N6-methyladenosine (m6A) is a ubiquitous feature of the mammalian transcriptome. It modulates mRNA fate and dynamics to exert regulatory control over numerous cellular processes and disease pathways, including viral infection. Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation from the latent phase leads to the redistribution of m6A topology upon both viral and cellular mRNAs within infected cells. Here we investigate the role of m6A in cellular transcripts upregulated during KSHV lytic replication. Our results show that m6A is crucial for the stability of the GPRC5A mRNA, whose expression is induced by the KSHV latent–lytic switch master regulator, the replication and transcription activator (RTA) protein. Moreover, we demonstrate that GPRC5A is essential for efficient KSHV lytic replication by directly regulating NFκB signalling. Overall, this work highlights the central importance of m6A in modulating cellular gene expression to influence viral infection

The Prp19 complex and the Usp4Sart3 deubiquitinating enzyme control reversible ubiquitination at the spliceosome

The spliceosome, a dynamic assembly of proteins and RNAs, catalyzes the excision of intron sequences from nascent mRNAs. Recent work has suggested that the activity and composition of the spliceosome are regulated by ubiquitination, but the underlying mechanisms have not been elucidated. Here, we report that the spliceosomal Prp19 complex modifies Prp3, a component of the U4 snRNP, with nonproteolytic K63-linked ubiquitin chains. The K63-linked chains increase the affinity of Prp3 for the U5 snRNP component Prp8, thereby allowing for the stabilization of the U4/U6.U5 snRNP. Prp3 is deubiquitinated by Usp4 and its substrate targeting factor, the U4/U6 recycling protein Sart3, which likely facilitates ejection of U4 proteins from the spliceosome during maturation of its active site. Loss of Usp4 in cells interferes with the accumulation of correctly spliced mRNAs, including those for α-tubulin and Bub1, and impairs cell cycle progression. We propose that the reversible ubiquitination of spliceosomal proteins, such as Prp3, guides rearrangements in the composition of the spliceosome at distinct steps of the splicing reaction

Biochemical purification of Drosophila casein kinase II.

<p>A) Purification strategy for endogenous casein kinase II. B) Protein composition of peak fraction of activity from the final heparin column visualized by SDS-PAGE and silver-staining. Species identified as casein kinase II alpha and beta are labeled. Bands labeled with an asterisk correspond to contaminating keratin. C) <i>In vitro</i> kinase assay of PSI mutant proteins. Serine to alanine PSI mutant proteins were phosphorylated <i>in vitro</i> using the peak fraction of activity from the final heparin column and using purified recombinant human casein kinase II (NEB P6010S). Assays were visualized using autoradiography, and, to ensure equal protein loading, coomassie staining.</p

Biochemcial fractionation and analysis of the PSI kinase.

<p>A) Purified recombinant PSI and PSI purified from Kc cells was treated with calf intestinal phosphatase (CIP) and then visualized by immunoblotting. B) MS2 spectra identifying phosphopeptides found in PSI. B and Y series ions and neutral loss of phosphate are indicated. Inset: sequence of the phosphopeptide and SEQUEST statistics. MS3 spectra and corresponding spectra of unmodified peptides are given in supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056401#pone-0056401-g001" target="_blank">Figure 1</a>.</p

Protein-protein interactions of PSI.

<p>A) GST pulldown assay using PSI mutant proteins. GST-PSI fusion proteins carrying the serine to alanine PSI mutations were phosphorylated using purified human casein kinase II and incubated with Kc nuclear extract. The resulting glutathione resin eluates were analyzed by silver staining and mass spectrometry. B) Silver stain of PSI and interacting proteins following anti-polyoma and anti-PSI immunoprecipitations. The asterisk indicates antibody heavy chain. C) Immunoblot analysis of (B) using anti-PSI antibody. D) Mass spectrometry analysis of (B). Proteins identified as interacting with PSI and the number of peptides observed for each protein are listed.</p