The eIF4E-binding protein Eap1p functions in Vts1p-mediated transcript decay.

Sequence-specific RNA binding proteins can induce the degradation of mRNAs through their ability to recruit proteins that trigger transcript destabilization. For example, Vts1p, the S. cerevisiae member of the Smaug family of RNA binding proteins, is thought to induce transcript decay by recruiting the Ccr4p-Pop2p-Not deadenylase complex to target mRNAs. The resulting deadenylation triggers transcript decapping followed by 5'-to-3' exonucleolytic decay. Here we show that the eIF4E-binding protein, Eap1p, is required for efficient degradation of Vts1p target transcripts and that this role involves the ability of Eap1p to interact with eIF4E. Eap1p does not stimulate deadenylation of Vts1p target transcripts but is instead involved in decapping. Eap1p interacts with Vts1p and mediates an indirect interaction between Vts1p and eIF4E. Taken together these data suggest a model whereby the interaction of Vts1p with Eap1p at target mRNAs stimulates decapping.

S. cerevisiae Vts1p induces deadenylation-dependent transcript degradation and interacts with the Ccr4p-Pop2p-Not deadenylase complex.

The Smaug family of sequence-specific RNA binding proteins regulates mRNA translation and degradation by binding to consensus stem-loop structures in target mRNAs. Vts1p is a member of the Smaug protein family that regulates the stability of target transcripts in Saccharomyces cerevisiae. Here we focus on the mechanism of Vts1p-mediated mRNA decay. Using RNA reporters that recapitulate Vts1p-mediated decay in vivo, we demonstrate that Vts1p stimulates mRNA degradation through deadenylation mediated by the Ccr4p-Pop2p-Not deadenylase complex. We also show that Vts1p interacts with the Ccr4p-Pop2p-Not complex suggesting that Vts1p recruits the Ccr4p-Pop2p-Not deadenylase complex to target mRNAs, resulting in transcript decay. Following deadenylation Vts1p target transcripts are decapped and subsequently degraded by the 5'-to-3' exonuclease Xrn1p. Decapping and 5'-to-3' decay is thought to occur in foci known as P-bodies, and we provide evidence that Vts1p function may involve P-bodies. Taken together with previous work, these data suggest that Smaug family members employ a conserved mechanism to induce transcript degradation that involves recruitment of the Ccr4-Pop2-Not deadenylase to target mRNAs.

The RNA-binding SAM domain of Smaug defines a new family of post-transcriptional regulators.

Anteroposterior patterning in Drosophila melanogaster is dependent on the sequence-specific RNA-binding protein Smaug, which binds to and regulates the translation of nanos (nos) mRNA. Here we demonstrate that the sterile-alpha motif (SAM) domain of Smaug functions as an RNA-recognition domain. This represents a new function for the SAM domain family, which is well characterized for mediating protein-protein interactions. Using homology modeling and site-directed mutagenesis, we have localized the RNA-binding surface of the Smaug SAM domain and have elaborated the RNA consensus sequence required for binding. Residues that compose the RNA-binding surface are conserved in a subgroup of SAM domain-containing proteins, suggesting that the function of the domain is conserved from yeast to humans. We show here that the SAM domain of Saccharomyces cerevisiae Vts1 binds RNA with the same specificity as Smaug and that Vts1 induces transcript degradation through a mechanism involving the cytoplasmic deadenylase CCR4. Together, these results suggest that Smaug and Vts1 define a larger class of post-transcriptional regulators that act in part through a common transcript-recognition mechanism.

Human kallikrein gene 5 (KLK5) expression by quantitative PCR: an independent indicator of poor prognosis in breast cancer.

BACKGROUND: KLK5 is a newly discovered human kallikrein gene. Many kallikrein genes have been found to be differentially expressed in various malignancies, and prostate-specific antigen (PSA; encoded by the KLK3 gene) is the best tumor marker for prostate cancer. Like the genes that encode PSA and other kallikreins, the KLK5 gene was found to be regulated by steroid hormones in the BT-474 breast cancer cell line. METHODS: We studied KLK5 expression in 179 patients with different stages and grades of epithelial breast carcinoma by quantitative reverse transcription-PCR (RT-PCR), using LightCycler((R)) technology. An optimal cutoff point equal to the detection limit (65th percentile) was used. KLK5 values were then compared with other established prognostic factors in terms of disease-free (DFS) and overall survival (OS). RESULTS: High KLK5 expression was found more frequently in pre-/perimenopausal (P = 0.026), node-positive (P = 0.029), and estrogen receptor-negative (P = 0.038) patients. In univariate analysis, KLK5 overexpression was a significant predictor of reduced DFS (P <0.001) and OS (P <0.001). Cox multivariate analysis indicated that KLK5 was an independent prognostic factor for DFS and OS. KLK5 remained an independent prognostic variable in the subgroups of patients with large tumors (>2 cm) and positive nodes. Hazard ratios derived from Cox analysis and related to DFS and OS were 2.48 (P = 0.005) and 2.37 (P = 0.009), respectively, for the node-positive group and 3.03 (P = 0.002) and 2.94 (P = 0.002), respectively, for patients with tumor sizes >2 cm. KLK5 expression was also associated with statistically significantly shorter DFS (P = 0.006) and OS (P = 0.004) in the subgroup of patients with grade I and II tumors. CONCLUSIONS: KLK5 expression as assessed by quantitative RT-PCR is an independent and unfavorable prognostic marker for breast carcinoma.

Down-regulation of the human kallikrein gene 5 (KLK5) in prostate cancer tissues.

BACKGROUND: Kallikreins are a subgroup of serine proteases with diverse physiological functions. Many kallikrein genes are differentially expressed in various malignancies and prostate specific antigen (PSA; encoded by the KLK3 gene) is the best tumor marker for prostate cancer. Human glandular kallikrein (hK2; encoded by the KLK2 gene) is an emerging tumor marker for prostate cancer. KLK5 is a newly discovered human kallikrein gene which shares a high degree of homology and is located adjacent to KLK2 and KLK3 genes on chromosome 19q13.4. Like KLK2 and KLK3, the KLK5 gene is regulated by steroid hormones in the BT-474 breast cancer cell line. We have previously shown that KLK5 is differentially expressed in ovarian and breast cancer. METHODS: We compared the expression of KLK5 in 29 pairs of histologically confirmed normal and prostate cancer tissues by quantitative RT-PCR using the LightCycler technology. RESULTS: KLK5 expression was significantly lower in cancer tissues compared to their normal counterparts. Lowest levels of expression were found in T3 stage tumors compared with T1 and T2. Also, a significant negative correlation was found between Gleason score and KLK5 expression. CONCLUSIONS: KLK5 should be further studied as a potential new prognostic marker in prostate cancer, whose expression is negatively correlated with cancer aggressiveness.