Missouri 65212 USA mRNAs R1 and R2 of the autonomous parvovirus minute virus of mice (MVM), which encode the viral nonstructural proteins NS1 and NS2, respectively, are processed in an ordered splicing pathway in which R2 is generated from mature spliced R1. Introduction of translation termination signals into these genes alters the processing of these RNAs; there is a significant (up to fourfold) increase in the accumulated steady-state levels of R1 relative to R2, when compared with wild-type levels, although the total accumulated levels of R1 plus R2 remain the same. The increase in accumulated R1 relative to R2 in mutant infected or transfected murine cells is independent of RNA stability and transport and decreases, in a polar manner, with the distance of the inserted termination signal from the shared initiation codon for NS1 and NS2 at nucleotide 260. The increased ratio of R1 to R2 is a consequence of the artificially introduced translation termination signals acting in cis rather than in the absence of a functional viral gene product. These mutations have an effect when they interrupt previously open reading frames in either exon of the spliced product R2. Nonsense mutations that are located in the second exon of R2 inhibit splicing of R1 to R2 only when they interrupt an open reading frame (ORF) that has the potential, after normal splicing, to be joined in-frame with the initiating AUG. These results suggest that nonsense mutations inhibit splicing of R1 to R2 by influencing the mechanism by which exons are defined in murine cells.

Stimulation of antigen receptors in T and B cells leads to the activation of the Src and Syk families of protein tyrosine kinases (PTK). These PTKs subsequently phosphorylate numerous intracellular substrates, including the 95-kD protooncogene product Vav. Vav is essential for both T and B cell development and T and B cell antigen receptor–mediated signal transduction. After receptor ligation, Vav associates with phosphorylated Syk and ZAP-70 PTKs, an interaction that depends upon its SH2 domain. Here we demonstrate that a point mutation of tyrosine 315 (Y315F) in ZAP-70, a putative Vav SH2 domain binding site, eliminated the Vav– ZAP-70 interaction. Moreover, the Y315 mutation impaired the function of ZAP-70 in antigen receptor signaling. Surprisingly, this mutation also resulted in marked reduction in the tyrosine phosphorylation of ZAP-70, Vav, SLP-76, and Shc. These data demonstrate that the Vav binding site in ZAP-70 plays a critical role in antigen receptor–mediated signal transduction.

The T-cell receptor (TCR) and B-cell receptor (BCR) are multimeric protein complexes consisting of antigen binding subunits (␣␤Ti for the TCR and membrane immunoglobulin [mIg] for the BCR) and the signal transducing invariant subunits (␥, ␦, and ε subunits of CD3 complex and proteins for the TCR; Ig␣ and Ig␤ for the BCR) (reviewed in reference 48). Engagement of the TCR and BCR initiates a cascade of intracellular processes that lead to cellular response. One of the earliest detectable biochemical events after TCR and BCR stimulation is the tyrosine phosphorylation of multiple cellular protein substrates, including the invariant chains of the TCR and BCR (reviewed in references 3, 4, 12, 29, 35, and 48).At least two families of protein tyrosine kinases (PTKs), the Src family and the Syk/ZAP-70 family, are implicated in regulating this tyrosine phosphorylation process (reviewed in reference 48). Both biochemical and genetic studies have demonstrated that Src family PTKs activated by TCR and BCR stimulation are required to phosphorylate the signal transducing subunits of these receptors (7,19,34,39). This phosphorylation occurs on the two tyrosine residues present in a common signaling motif which exists as one copy in CD3␥, CD3␦, CD3ε, Ig␣, and Ig␤ and as three copies in TCR (33). This motif, the immunoreceptor tyrosine-based activation motif (ITAM), consists of pairs of tyrosine and leucine residues arranged in the consensus sequence YxxL(x) 6-8 YxxL, where x is variable. After TCR stimulation, ZAP-70 is recruited to the receptor complex through the interaction of its two SH2 domains with the doubly phosphorylated ITAMs (19,46). This interaction is believed to be critical for TCR signaling, since phosphopeptides that block the interaction of ZAP-70 with the chain also inhibit TCR signaling events (45). The association of ZAP-70 with the TCR ITAMs facilitates its autophosphorylation and the tyrosine phosphorylation of ZAP-70 mediated by Src family PTKs (19,27). The critical role for ZAP-70 in T-cell, not B-cell, activation and development has been demonstrated in patients with severe combined immunodeficiency who were deficient in 8,15) and in mice which had been made deficient in . Similarly, a critical role for Syk in B-cell, not ␣␤ T-cell, activation and development has been shown both in chicken B cells (39) and in mice which had been made deficient in Syk (9, 41).Like Syk, ZAP-70 is composed of three easily identifiable domains, a tyrosine kinase domain and two tandemly arranged SH2 domains (N terminal and C terminal) which mediate the association of ZAP-70 with the TCR after its stimulation (7,19,46). Between the two SH2 domains exists a region (60 amino acids [aa] in length; interdomain A) which forms a coiled-coil structure and is likely involved in bringing together the two SH2 domains which bind to the receptor ITAMs (18). Between the second SH2 domain and the kinase domain lies an additional region (84 aa in size; interdomain B) whose structure and function are unclear. Interdomain B contains a prolin...

Background and Aims Nonalcoholic fatty liver disease (NAFLD) is characterized by accumulation of excessive triglycerides (TGs) in hepatocytes. Obesity is a major risk factor for developing fatty liver, although the intracellular molecular basis remains largely unclear. N6‐methyladenosine (m6A) RNA methylation is the most common internal modification in eukaryotic mRNA. Approach and Results In the present study, by m6A sequencing and RNA sequencing, we found that both m6A enrichment and mRNA expression of lipogenic genes were significantly increased in leptin‐receptor–deficient db/db mice. Importantly, our results showed that YT521‐B homology domain‐containing 2 (Ythdc2), an m6A reader, was markedly down‐regulated in livers of obese mice and NAFLD patients. Suppression of Ythdc2 in livers of lean mice led to TG accumulation, whereas ectopic overexpression of Ythdc2 in livers of obese mice improved liver steatosis and insulin resistance. Mechanistically, we found that Ythdc2 could bind to mRNA of lipogenic genes, including sterol regulatory element‐binding protein 1c, fatty acid synthase, stearoyl‐CoA desaturase 1, and acetyl‐CoA carboxylase 1, to decrease their mRNA stability and inhibit gene expression. Conclusions Our findings describe an important role of the m6A reader, Ythdc2, for regulation of hepatic lipogenesis and TG homeostasis, which might provide a potential target for treating obesity‐related NAFLD.

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High expression of cancer stem cell (CSC) markers is related to poor prognosis of patients with hepatocellular carcinoma (HCC). However, the expression of these markers in patient-derived xenograft (PDX) models and the relationship of the expression levels of these markers between HCC patients and their PDX models at subsequent low passages are unclear. To investigate the prognostic impact of putative CSC markers in patients with HCC and in related PDX models, the expression of CD133, CD90, CD44, ALDH1, CK7, CK19, OCT4, SOX2, vimentin, nestin, CD13 and EpCam were assessed by quantitative reverse transcription-PCR (qRT-PCR) and then were validated using immunohistochemistry in tumor or peritumoral tissues from patients and tumor tissues from PDX models. Cumulative survival analysis of the patients and animals was conducted using the Kaplan-Meier method and the log-rank test. Only the expression levels of CD133 and CD44 were higher in tumor tissues than in the peritumoral tissues of HCC patients by qRT-PCR. High consistency of the prognostic value of the expression of CD133/CD44 was observed in HCC patients and the PDX models. High expression levels of CD133 and CD44 were positively related to the poor prognosis of HCC patients and to that in the PDX models. PDX HCC models in the present study have been suggested to be predictive of disease outcome, which could shed light on personalized medicine and the mechanisms of CSC marker expression on prognosis.