The Puf family of RNA-binding proteins regulates gene expression primarily by interacting with the 3′ untranslated region (3′ UTR) of targeted mRNAs and inhibiting translation and/or stimulating decay. Physical association and computational analyses of yeast Puf3p identified >150 potential mRNA targets involved in mitochondrial function. However, only COX17 has been established as a target of Puf3p-mediated deadenylation and decapping. We have identified 10 new targets that are rapidly degraded in a Puf3p-dependent manner. We also observed changes in Puf3p activity in response to environmental conditions. Puf3p promotes rapid degradation of mRNA targets in the fermentable carbon source dextrose. However, Puf3p-mediated decay activity is inhibited in carbon sources that require mitochondrial function for efficient cell growth. In addition, the activity of Puf3p is rapidly altered by changing the carbon source. PUF3 expression is not decreased at the RNA or protein level by different carbon sources and localization is not significantly altered, suggesting that Puf3p activity is regulated posttranslationally. Finally, under conditions when Puf3p is unable to stimulate decay, Puf3p can still bind its target mRNAs. Together, these experiments provide insight into the carbon source-specific control of Puf3p activity and how such alterations allow Puf3p to dynamically regulate mitochondrial function.

Cells must make careful use of the resources available to them. A key area of cellular regulation involves the biogenesis of ribosomes. Transcriptional regulation of ribosome biogenesis factor genes through alterations in histone acetylation has been well studied. This work identifies a post-transcriptional mechanism of ribosome biogenesis regulation by Puf protein control of mRNA stability. Puf proteins are eukaryotic mRNA binding proteins that play regulatory roles in mRNA degradation and translation via association with specific conserved elements in the 3ʹ untranslated region (UTR) of target mRNAs and with degradation and translation factors. We demonstrate that several ribosome biogenesis factor mRNAs in Saccharomyces cerevisiae containing a canonical Puf4p element in their 3ʹ UTRs are destabilized by Puf2p, Puf4, and Puf5p, yet stabilized by Puf1p and Puf3p. In the absence of all Puf proteins, these ribosome biogenesis mRNAs are destabilized by a secondary mechanism involving the same 3ʹ UTR element. Unlike other targets of Puf4p regulation, the decay of these transcripts is not altered by carbon source. Overexpression of Puf4p results in delayed ribosomal RNA processing and altered ribosomal subunit trafficking. These results represent a novel role for Puf proteins in yeast as regulators of ribosome biogenesis transcript stability.

Human genetic studies have identified a large number of disease-associated de novo variants in presumptive regulatory regions of the genome that pose a challenge for interpretation of their effects: the impact of regulatory variants is highly dependent on the cellular context, and thus for psychiatric diseases these would ideally be studied in neurons in a living brain. Furthermore, for both common and rare variants, it is expected that only a subset fraction will affect gene expression. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences in a single experiment. These assays have been used for functional screening of several different types of regulatory sequences in vitro. However, they have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3′UTR MPRA library with variants from ASD patients, we sought to develop a method to achieve reproducible measurements of variant effects in vivo in a cell type-specific manner. We implemented a Cre-dependent design to control expression of our library and first validated our system in vitro. Next, we measured the effect of >500 3′UTR variants in excitatory neurons in the mouse brain. Finally, we report >40 variants with significant effects on transcript abundance in the context of the brain. This new technique should enable robust, functional annotation of genetic variants in the cellular contexts most relevant to psychiatric disease.

Colorectal cancer (CRC) tumorigenesis and progression are linked to common oncogenic mutations, especially in the tumor suppressor APC, whose loss triggers the deregulation of TCF4/β-Catenin activity. CRC tumorigenesis is also driven by multiple epi-mutational modifiers, such as transcriptional regulators. We describe the common (and near-universal) activation of the zinc finger transcription factor and Let-7 target PLAGL2 in CRC and find that it is a key driver of intestinal epithelial transformation. PLAGL2 drives proliferation, cell cycle progression, and anchorage-independent growth in CRC cell lines and non-transformed intestinal cells. Investigating effects of PLAGL2 on downstream pathways revealed very modest effects on canonical Wnt signaling. Alternatively, we find pronounced effects on the direct PLAGL2 target genes IGF2, a fetal growth factor, and ASCL2, an intestinal stem cell-specific bHLH transcription factor. Inactivation of PLAGL2 in CRC cell lines has pronounced effects on ASCL2 reporter activity. Furthermore, ASCL2 expression can partially rescue deficits of proliferation and cell cycle progression caused by depletion of PLAGL2 in CRC cell lines. Thus, the oncogenic effects of PLAGL2 appear to be mediated via core stem cell and onco-fetal pathways, with minimal effects on downstream Wnt signaling.

Colorectal cancer (CRC) tumorigenesis and progression are linked to common oncogenic mutations, especially in the tumor suppressor APC, whose loss triggers the deregulation of TCF4/β-Catenin activity. CRC tumorigenesis is also driven by multiple epi-mutational modifiers, such as transcriptional regulators. We describe the common (and near-universal) activation of the zinc finger transcription factor and Let-7 target PLAGL2 in CRC and find that it is a key driver of intestinal epithelial transformation. PLAGL2 drives proliferation, cell cycle progression, and anchorage-independent growth in CRC cell lines and non-transformed intestinal cells. Investigating effects of PLAGL2 on downstream pathways revealed very modest effects on canonical Wnt signaling. Alternatively, we find pronounced effects on the direct PLAGL2 target genes IGF2, a fetal growth factor, and ASCL2, an intestinal stem cell-specific bHLH transcription factor. Inactivation of PLAGL2 in CRC cell lines has pronounced effects on ASCL2 reporter activity. Furthermore, ASCL2 expression can partially rescue deficits of proliferation and cell cycle progression caused by depletion of PLAGL2 in CRC cell lines. Thus, the oncogenic effects of PLAGL2 appear to be mediated via core stem cell and onco-fetal pathways, with minimal effects on downstream Wnt signaling.