Specific sequences within arginine–glycine-rich domains affect mRNA-binding protein function

The discovery of roles for arginine methylation in intracellular transport and mRNA splicing has focused attention on the methylated arginine–glycine (RG)-rich domains found in many eukaryotic RNA-binding proteins. Sequence similarity among these highly repetitive RG domains, combined with interactions between RG-rich proteins, raises the question of whether these regions are general interaction motifs or whether there is specificity within these domains. Using the essential Saccharomyces cerevisiae mRNA-binding protein Npl3 (ScNpl3) as a model system, we first tested the importance of the RG domain for protein function. While Npl3 lacking the RG domain could not support growth of cells lacking Npl3, surprisingly, expression of the RG domain alone supported partial growth of these cells. To address the specificity of this domain, we created chimeric forms of ScNpl3 with RG-rich domains of S. cerevisiae nucleolar proteins, Gar1 and Nop1 (ScGar1, ScNop1), or of the Candida albicans Npl3 ortholog (CaNpl3). Whereas the CaNpl3 RG chimeric protein retained nearly wild-type function in S. cerevisiae, the ScGar1 and ScNop1 RG domains significantly reduced Npl3 function and self-association, indicating RG domain specificity. Nuclear localization of Npl3 also requires specific RG sequences, yet heterologous RG domains allow similar modulation of Npl3 transport by arginine methylation

Individual influenza A virus mRNAs show differential dependence on cellular NXF1/TAP for their nuclear export

The influenza A virus RNA-dependent RNA polymerase produces capped and polyadenylated mRNAs in the nucleus of infected cells that resemble mature cellular mRNAs, but are made by very different mechanisms. Furthermore, only two of the 10 viral protein-coding mRNAs are spliced: most are intronless, while two contain unremoved introns. The mechanism(s) by which any of these mRNAs are exported from the nucleus is uncertain. To probe the involvement of the primary cellular mRNA export pathway, we treated cells with siRNAs against NXF1, Aly or UAP56, or with the drug 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), an inhibitor of RNA polymerase II phosphorylation previously shown to inhibit nuclear export of cellular mRNA as well as influenza virus segment 7 mRNAs. Depletion of NXF1 or DRB treatment had similar effects, inhibiting the nuclear export of several of the viral mRNAs. However, differing degrees of sensitivity were seen, depending on the particular segment examined. Intronless HA mRNA and spliced M2 or unspliced M1 transcripts (all encoding late proteins) showed a strong requirement for NXF1, while intronless early gene mRNAs, especially NP mRNA, showed the least dependency. Depletion of Aly had little effect on viral mRNA export, but reduction of UAP56 levels strongly inhibited trafficking and/or translation of the M1, M2 and NS1 mRNAs. Synthesis of NS2 from the spliced segment 8 transcript was, however, resistant. We conclude that influenza A virus co-opts the main cellular mRNA export pathway for a subset of its mRNAs, including most but not all late gene transcripts

Genetic and biochemical analysis of the serine/threonine protein kinases PknA, PknB, PknG and PknL of Corynebacterium glutamicum: evidence for non-essentiality and for phosphorylation of OdhI and FtsZ by multiple kinases

We previously showed that the 2-oxoglutarate dehydrogenase inhibitor protein OdhI of Corynebacterium glutamicum is phosphorylated by PknG at Thr14, but that also additional serine/threonine protein kinases (STPKs) can phosphorylate OdhI. To identify these, a set of three single (ΔpknA, ΔpknB, ΔpknL), five double (ΔpknAG, ΔpknAL, ΔpknBG, ΔpknBL, ΔpknLG) and two triple deletion mutants (ΔpknALG, ΔpknBLG) were constructed. The existence of these mutants shows that PknA, PknB, PknG and PknL are not essential in C. glutamicum. Analysis of the OdhI phosphorylation status in the mutant strains revealed that all four STPKs can contribute to OdhI phosphorylation, with PknG being the most important one. Only mutants in which pknG was deleted showed a strong growth inhibition on agar plates containing glutamine as carbon and nitrogen source. Thr14 and Thr15 of OdhI were shown to be phosphorylated in vivo, either individually or simultaneously, and evidence for up to two additional phosphorylation sites was obtained. Dephosphorylation of OdhI was shown to be catalysed by the phospho-Ser/Thr protein phosphatase Ppp. Besides OdhI, the cell division protein FtsZ was identified as substrate of PknA, PknB and PknL and of the phosphatase Ppp, suggesting a role of these proteins in cell division

Cotranscriptional recruitment of the nuclear poly(A)-binding protein Pab2 to nascent transcripts and association with translating mRNPs

Synthesis of the pre-mRNA poly(A) tail in the nucleus has important consequences on the translational activity of the mature mRNA in the cytoplasm. In most eukaryotes, nuclear polyadenylation of pre-mRNAs is thought to require the nuclear poly(A)-binding protein (PABP2/PABPN1) for poly(A) tail synthesis and ultimate length control. As yet, however, the extent of the association between PABP2 and the exported mRNA remains poorly understood. Here, we used chromatin immunoprecipitation (ChIP) assays to show that the fission yeast ortholog of mammalian PABP2 (Pab2) is cotranscriptionally recruited to active genes. Notably, the association of Pab2 to genes precedes that of a typical 3′-processing/polyadenylation factor, suggesting that Pab2 recruitment during the transcription cycle precedes polyadenylation. The inclusion of an RNase step in our ChIP and immunoprecipitation assays suggests that Pab2 is cotranscriptionally recruited via nascent mRNA ribonucleoprotein (mRNPs). Tandem affinity purification coupled with mass spectrometry also revealed that Pab2 associates with several ribosomal proteins as well as general translation factors. Importantly, whereas previous results suggest that the nuclear poly(A)-binding protein is not present on cytoplasmic mRNAs, we show that fission yeast Pab2 is associated with polysomes. Our findings suggest that Pab2 is recruited to nascent mRNPs during transcription and remains associated with translated mRNPs after nuclear export

Exon junction complexes mediate the enhancing effect of splicing on mRNA expression

Intron-containing genes are generally expressed more effectively in human cells than are intronless versions of the same gene. We have asked whether this effect is due directly to splicing or instead reflects the action of components of the exon junction complex (EJC) that is assembled at splice junctions after splicing is completed. Here, we show that intron removal does not enhance gene expression if EJC formation is blocked. Conversely, RNA tethering of the EJC components SRm160 or RNPS1 boosts the expression of intronless mRNAs but not of spliced mRNAs. Splicing and RNPS1 tethering are shown to enhance the same steps in mRNA biogenesis and function, including mRNA 3′ end processing and translation. Together, these data argue that the EJC is primarily responsible for the positive effect of splicing on gene expression

Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method

Detailed knowledge of the composition and structure of the spliceosome and its assembly intermediates is a prerequisite for understanding the complex process of pre-mRNA splicing. To this end, we have developed a tobramycin affinity-selection method that is generally applicable for the purification of native RNP complexes. By using this method, we have isolated human prespliceosomes that are ideally suited for both biochemical and structural studies. MS identified >70 prespliceosome-associated proteins, including nearly all known U1 and U2 snRNP proteins, and expected non-snRNP splicing factors. In addition, the DEAD-box protein p68, RNA helicase A, and a number of proteins that appear to perform multiple functions in the cell, such as YB-1 and TLS, were detected. Several previously uncharacterized proteins of unknown function were also identified, suggesting that they play a role in splicing and potentially act during prespliceosome assembly. These data provide insight into the complexity of the splicing machinery at an early stage of its assembly

Splicing of U12-type introns deposits an exon junction complex competent to induce nonsense-mediated mRNA decay

Metazoan cells have two pathways for intron removal involving the U2- and U12-type spliceosomes, which contain mostly nonoverlapping sets of small nuclear ribonucleoproteins. We show that in vitro splicing of a U12-type intron assembles an exon junction complex (EJC) that is comparably positioned and contains many of the same components as that deposited by the U2-type spliceosome. The presence of a U12-type intron downstream of a premature termination codon within an open reading frame (ORF) induces nonsense-mediated decay of the mRNA in vivo. These findings suggest a common pathway for EJC assembly by the two spliceosomes and highlight the evolutionary age of the EJC and its downstream functions in gene expression

Role of purine-rich exonic splicing enhancers in nuclear retention of pre-mRNAs

Intron-containing pre-mRNAs are normally retained in the nucleus until they are spliced to produce mature mRNAs that are exported to the cytoplasm. Although the detailed mechanism is not well understood, the formation of splicing-related complexes on pre-mRNAs is thought to be responsible for the nuclear retention. Therefore, pre-mRNAs containing suboptimal splice sites should tend to leak out to the cytoplasm. Such pre-mRNAs often contain purine-rich exonic splicing enhancers (ESEs) that stimulate splicing of the adjacent intron. Here, we show that ESEs per se possess an activity to retain RNAs in the nucleus through a saturable nuclear retention factor. Cross-competition experiments revealed that intron-containing pre-mRNAs (without ESEs) used the same saturable nuclear retention factor as ESEs. Interestingly, although intronless mRNAs containing ESEs were also poorly exported, spliced mRNAs produced from ESE-containing pre-mRNAs were efficiently exported to the cytoplasm. Thus, the splicing reaction can reset the nuclear retention state caused by ESEs, allowing nuclear export of mature mRNAs. Our results reveal a novel aspect of ESE activity that should contribute to gene expression and RNA quality control

Genes with internal repeats require the THO complex for transcription

The evolutionarily conserved multisubunit THO complex, which is recruited to actively transcribed genes, is required for the efficient expression of FLO11 and other yeast genes that have long internal tandem repeats. FLO11 transcription elongation in Tho(−) mutants is hindered in the region of the tandem repeats, resulting in a loss of function. Moreover, the repeats become genetically unstable in Tho(−) mutants. A FLO11 gene without the tandem repeats is transcribed equally well in Tho(+) or Tho(−) strains. The Tho(−) defect in transcription is suppressed by overexpression of topoisomerase I, suggesting that the THO complex functions to rectify aberrant structures that arise during transcription

Crystal structure of the archaeal ammonium transporter Amt-1 from Archaeoglobus fulgidus

Ammonium transporters (Amts) are integral membrane proteins found in all kingdoms of life that fulfill an essential function in the uptake of reduced nitrogen for biosynthetic purposes. Amt-1 is one of three Amts encoded in the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus. The crystal structure of Amt-1 shows a compact trimer with 11 transmembrane helices per monomer and a central channel for substrate conduction in each monomer, similar to the known crystal structure of AmtB from Escherichia coli. Xenon derivatization has been used to identify apolar regions of Amt-1, emphasizing not only the hydrophobicity of the substrate channel but also the unexpected presence of extensive internal cavities that should be detrimental for protein stability. The substrates ammonium and methylammonium have been used for cocrystallization experiments with Amt-1, but the identification of binding sites that are distinct from water positions is not unambiguous. The well ordered cytoplasmic C terminus of the protein in the Amt-1 structure has allowed for the construction of a docking model between Amt-1 and a homology model for its physiological interaction partner, the P(II) protein GlnB-1. In this model, GlnB-1 binds tightly to the cytoplasmic face of the transporter, effectively blocking conduction through the three individual substrate channels