Facilitated leaky scanning and atypical ribosome shunting direct downstream translation initiation on the tricistronic S1 mRNA of avian reovirus

The S1 mRNA of avian reovirus is functionally tricistronic, encoding three unrelated proteins, p10, p17 and σC, from three sequential, partially overlapping open reading frames (ORFs). The mechanism of translation initiation at the 3′-proximal σC ORF is currently unknown. Transient RNA transfections using Renilla luciferase reporter constructs revealed only a modest reduction in reporter expression upon optimization of either the p10 or p17 start sites. Insertion of multiple upstream AUG (uAUG) codons in a preferred start codon sequence context resulted in a substantial retention of downstream translation initiation on the S1 mRNA, but not on a heterologous mRNA. The S1 mRNA therefore facilitates leaky scanning to promote ribosome access to the σC start codon. Evidence also indicates that σC translation is mediated by a second scanning-independent mechanism capable of bypassing upstream ORFs. This alternate mechanism is cap-dependent and requires a sequence-dependent translation enhancer element that is complementary to 18S rRNA. Downstream translation initiation of the tricistronic S1 mRNA is therefore made possible by two alternate mechanisms, facilitated leaky scanning and an atypical form of ribosome shunting. This dual mechanism of downstream translation initiation ensures sufficient expression of the σC cell attachment protein that is essential for infectious progeny virus production

Soybean ENOD40 encodes two peptides that bind to sucrose synthase

ENOD40 is expressed at an early stage in root nodule organogenesis in legumes. Identification of ENOD40 homologs in nonleguminous plants suggests that this gene may have a more general biological function. In vitro translation of soybean ENOD40 mRNA in wheat germ extracts revealed that the conserved nucleotide sequence at the 5′ end (region I) encodes two peptides of 12 and 24 aa residues (peptides A and B). These peptides are synthesized de novo from very short, overlapping ORFs. Appropriate ORFs are present in all legume ENOD40s studied thus far. In this case small peptides are directly translated from polycistronic eukaryotic mRNA. The 24-aa peptide B was detected in nodules by Western blotting. Both peptides specifically bind to the same 93-kDa protein, which was affinity purified from soybean nodules. Using peptide mass fingerprinting, we identified this binding protein as nodulin 100, which is a subunit of sucrose synthase. Based on our data we suggest that ENOD40 peptides are involved in the control of sucrose use in nitrogen-fixing nodules

An unusual internal ribosomal entry site of inverted symmetry directs expression of a potato leafroll polerovirus replication-associated protein

Potato leafroll polerovirus (PLRV) genomic RNA acts as a polycistronic mRNA for the production of proteins P0, P1, and P2 translated from the 5′-proximal half of the genome. Within the P1 coding region we identified a 5-kDa replication-associated protein 1 (Rap1) essential for viral multiplication. An internal ribosome entry site (IRES) with unusual structure and location was identified that regulates Rap1 translation. Core structural elements for internal ribosome entry include a conserved AUG codon and a downstream GGAGAGAGAGG motif with inverted symmetry. Reporter gene expression in potato protoplasts confirmed the internal ribosome entry function. Unlike known IRES motifs, the PLRV IRES is located completely within the coding region of Rap1 at the center of the PLRV genome

Syk is a dual-specificity kinase that self-regulates the signal output from the B-cell antigen receptor

Upon B-cell activation, the signaling subunits Ig-α and Ig-β of the B-cell antigen receptor become phosphorylated not only on tyrosines but also on serine residues. Using a specific antibody, we show that serine 197 (S197) in the cytoplasmic tail of Ig-α is phosphorylated upon B-cell antigen receptor activation, and that this modification inhibits the signal output of the B-cell antigen receptor. Surprisingly, we found that the well-known protein tyrosine kinase Syk (spleen tyrosine kinase) phosphorylates S197 on Ig-α, thus not only activating but also inhibiting signaling from the B-cell antigen receptor. This finding identifies Syk as a dual-specificity kinase and establishes a previously unexplored paradigm for the self-regulation of biological signaling processes

Prostate and lymph node proton magnetic resonance (MR) spectroscopic imaging with external array coils at 3 T to detect recurrent prostate cancer after radiation therapy.

Contains fulltext : 53162.pdf (publisher's version ) (Closed access)In a patient suspected of having recurrent prostate cancer after radiation therapy, we demonstrate the feasibility of noninvasive proton magnetic resonance spectroscopic (1H-MRS) imaging of the prostate and a lymph node at 3 T using a matrix of external surface coils. Written informed consent was obtained from the patient. With 1H-MRS imaging, high choline with low citrate signal was observed in the prostate, and in the lymph node a signal of choline-containing compounds was identified. The tissue level of the compounds in the enlarged lymph node was estimated to be 8.1 mmol/kg water. Subsequent histopathological analysis of systematic transrectal ultrasound-guided prostate biopsy and computed tomography-guided biopsy of the lymph node confirmed the presence of prostate cancer in both

Tyr130 phosphorylation triggers Syk release from antigen receptor by long-distance conformational uncoupling

The Syk protein-tyrosine kinase plays a major role in signaling through the B cell receptor for antigen (BCR). Syk binds the receptor via its tandem pair of SH2 domains interacting with a doubly phosphorylated immunoreceptor tyrosine-based activation motif (dp-ITAM) of the BCR complex. Upon phosphorylation of Tyr-130, which lies between the two SH2 domains distant to the phosphotyrosine binding sites, Syk dissociates from the receptor. To understand the structural basis for this dissociation, we investigated the structural and dynamic characteristics of the wild type tandem SH2 region (tSH2) and a variant tandem SH2 region (tSH2pm) with Tyr-130 substituted by Glu to permanently introduce a negative charge at this position. NMR heteronuclear relaxation experiments, residual dipolar coupling measurements and analytical ultracentrifugation revealed substantial differences in the hydrodynamic behavior of tSH2 and tSH2pm. Although the two SH2 domains in tSH2 are tightly associated, the two domains in tSH2pm are partly uncoupled and tumble in solution with a faster correlation time. In addition, the equilibrium dissociation constant for the binding of tSH2pm to dp-ITAM (1.8 μM) is significantly higher than that for the interaction between dp-ITAM and tSH2 but is close to that for a singly tyrosine-phosphorylated peptide binding to a single SH2 domain. Experimental data and hydrodynamic calculations both suggest a loss of domain-domain contacts and change in relative orientation upon the introduction of a negative charge on residue 130. A long-distance structural mechanism by which the phosphorylation of Y130 negatively regulates the interaction of Syk with immune receptors is proposed

Epstein–Barr virus latent infection membrane protein 1 TRAF-binding site induces NIK/IKKα-dependent noncanonical NF-κB activation

Epstein–Barr virus (EBV) latent infection membrane protein 1 (LMP1)-induced NF-κB activation is important for infected cell survival. LMP1 activates NF-κB, in part, by engaging tumor necrosis factor (TNF) receptor-associated factors (TRAFs), which also mediate NF-κB activation from LTβR and CD40. LTβR and CD40 activation of p100/NF-κB2 is now known to be NIK/IKKα-dependent and IKKβ/IKKγ independent. In the experiments described here, we found that EBV LMP1 induced p100/NF-κB2 processing in human lymphoblasts and HEK293 cells. LMP1-induced p100 processing was NIK/IKKα dependent and IKKβ/IKKγ independent. Furthermore, the LMP1 TRAF-binding site was required for p100 processing and p52 nuclear localization, whereas the LMP1 death domain-binding site was not. Moreover, the LMP1 TRAF-binding site preferentially caused RelB nuclear accumulation. In murine embryo fibroblasts (MEFs), IKKβ was essential for LMP1 up-regulation of macrophage inflammatory protein (MIP)-2, TNFα, I-TAC, ELC, MIG, and CXCR4 RNAs. Interestingly, in IKKα knockout MEFs, LMP1 hyperinduced MIP-2, TNFα, and I-TAC expression, consistent with a role for IKKα in down-modulating canonical IKKβ activation or its effects. In contrast, LMP1 failed to up-regulate CXCR4 and MIG RNA in IKKα knockout MEFs, indicating a dependence on noncanonical IKKα activation. Furthermore, LMP1 up-regulation of MIP-2 RNA in MEFs was both IKKβ- and IKKγ-dependent, whereas LMP1 upregulation of MIG and I-TAC RNA was fully IKKγ independent. Thus, LMP1 induces typical canonical IKKβ/IKKγ-dependent, atypical canonical IKKβ-dependent/IKKγ-independent, and noncanonical NIK/IKKα-dependent NF-κB activations; NIK/IKKα-dependent NF-κB activation is principally mediated by the LMP1 TRAF-binding site

A unique lymphotoxin αβ-dependent pathway regulates thymic emigration of Vα14 invariant natural killer T cells

Natural killer (NK) T cells using an invariant Vα14 (Vα14i) T cell receptor rearrangement form a distinct immunoregulatory T cell lineage. Several studies indicated that a NK1.1(−) Vα14i NKT precursor cell differentiates and expands within the thymus before export to the peripheral tissues occurs. However, little is known about the signals that cause the emigration of Vα14i NKT cells from the thymus to the periphery. Here we show that signaling of lymphotoxin (LT) αβ through the LTβ receptor (LTβR) is indispensable for regulating peripheral but not thymic Vα14i NKT cell numbers. Homing to and homeostatic proliferation of thymic Vα14i NKT cells in peripheral organs, however, was not dependent on LTβR. Instead, our data indicate that a LTβR-expressing thymic stromal cell regulates the thymic emigration of Vα14i NKT cells but not conventional T cell receptor αβ cells