Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation.

Poly(A)-binding protein (PABP) stimulates translation initiation by binding simultaneously to the mRNA poly(A) tail and eukaryotic translation initiation factor 4G (eIF4G). PABP activity is regulated by PABP-interacting (Paip) proteins. Paip1 binds PABP and stimulates translation by an unknown mechanism. Here, we describe the interaction between Paip1 and eIF3, which is direct, RNA independent, and mediated via the eIF3g (p44) subunit. Stimulation of translation by Paip1 in vivo was decreased upon deletion of the N-terminal sequence containing the eIF3-binding domain and upon silencing of PABP or several eIF3 subunits. We also show the formation of ternary complexes composed of Paip1-PABP-eIF4G and Paip1-eIF3-eIF4G. Taken together, these data demonstrate that the eIF3-Paip1 interaction promotes translation. We propose that eIF3-Paip1 stabilizes the interaction between PABP and eIF4G, which brings about the circularization of the mRNA.

Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation.

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3' poly(A) tail-PABP complex.

A COMPARISON OF TWO METHODS FOR THE DETECTION OF FECAL POLLUTION IN DRINKING WATER (HYDROGEN SULFIDE, INDICATOR, MODIFICATION)

A new technique for the detection of microbiological fecal pollution in drinking and in raw surface water has been modified and tested against the standard multiple-tube fermentation technique (most-probable-number, MPN). The performance of the new test in detecting fecal pollution in drinking water has been tested at different incubation temperatures. The basis for the new test was the detection of hydrogen sulfide produced by the hydrogen sulfide producing bacteria which are usually associated with the coliform group. The positive results are indicated by the appearance of a brown to black color in the contents of the fermentation tube within 18 to 24 hours of incubation at 35 (+OR-) .5(DEGREES)C. For this study 158 water samples of different sources have been used. The results were analyzed statistically with the paired t-test and the one-way analysis of variance. No statistically significant difference was noticed between the two methods, when tested 35 (+OR-) .5(DEGREES)C, in detecting fecal pollution in drinking water. The new test showed more positive results with raw surface water, which could be due to the presence of hydrogen sulfide producing bacteria of non-fecal origin like Desulfovibrio and Desulfomaculum. The survival of the hydrogen sulfide producing bacteria and the coliforms was also tested over a 7-day period, and the results showed no significant difference. The two methods showed no significant difference when used to detect fecal pollution at a very low coliform density. The results showed that the new test is mostly effective, in detecting fecal pollution in drinking water, when used at 35 (+OR-) .5(DEGREES)C. The new test is effective, simple, and less expensive when used to detect fecal pollution in drinking water and raw surface water at 35 (+OR-) .5(DEGREES)C. The method can be used for qualitative and/or quantitative analysis of water in the field and in the laboratory. ^