Comparison of software packages for detecting unannotated translated small open reading frames by Ribo-seq.

Accurate and comprehensive annotation of microprotein-coding small open reading frames (smORFs) is critical to our understanding of normal physiology and disease. Empirical identification of translated smORFs is carried out primarily using ribosome profiling (Ribo-seq). While effective, published Ribo-seq datasets can vary drastically in quality and different analysis tools are frequently employed. Here, we examine the impact of these factors on identifying translated smORFs. We compared five commonly used software tools that assess open reading frame translation from Ribo-seq (RibORFv0.1, RibORFv1.0, RiboCode, ORFquant, and Ribo-TISH) and found surprisingly low agreement across all tools. Only ~2% of smORFs were called translated by all five tools, and ~15% by three or more tools when assessing the same high-resolution Ribo-seq dataset. For larger annotated genes, the same analysis showed ~74% agreement across all five tools. We also found that some tools are strongly biased against low-resolution Ribo-seq data, while others are more tolerant. Analyzing Ribo-seq coverage revealed that smORFs detected by more than one tool tend to have higher translation levels and higher fractions of in-frame reads, consistent with what was observed for annotated genes. Together these results support employing multiple tools to identify the most confident microprotein-coding smORFs and choosing the tools based on the quality of the dataset and the planned downstream characterization experiments of the predicted smORFs.

The landscape of fetus metabolism in maternal hyperglycemia.

Maternal hyperglycemia contributes to abnormal fetal development; yet, how it affects fetal metabolism is poorly understood. Perez-Ramirez and colleagues recently provided a comprehensive metabolic atlas of fetal organs isolated from normal and diabetic pregnant mice, identifying novel metabolites and alterations in tissue glucose utilization throughout mid-to-late gestation by maternal hyperglycemia.

The TINCR ubiquitin-like microprotein is a tumor suppressor in squamous cell carcinoma.

The TINCR (Terminal differentiation-Induced Non-Coding RNA) gene is selectively expressed in epithelium tissues and is involved in the control of human epidermal differentiation and wound healing. Despite its initial report as a long non-coding RNA, the TINCR locus codes for a highly conserved ubiquitin-like microprotein associated with keratinocyte differentiation. Here we report the identification of TINCR as a tumor suppressor in squamous cell carcinoma (SCC). TINCR is upregulated by UV-induced DNA damage in a TP53-dependent manner in human keratinocytes. Decreased TINCR protein expression is prevalently found in skin and head and neck squamous cell tumors and TINCR expression suppresses the growth of SCC cells in vitro and in vivo. Consistently, Tincr knockout mice show accelerated tumor development following UVB skin carcinogenesis and increased penetrance of invasive SCCs. Finally, genetic analyses identify loss-of-function mutations and deletions encompassing the TINCR gene in SCC clinical samples supporting a tumor suppressor role in human cancer. Altogether, these results demonstrate a role for TINCR as protein coding tumor suppressor gene recurrently lost in squamous cell carcinomas.

Profiling mouse brown and white adipocytes to identify metabolically relevant small ORFs and functional microproteins.

Microproteins (MPs) are a potentially rich source of uncharacterized metabolic regulators. Here, we use ribosome profiling (Ribo-seq) to curate 3,877 unannotated MP-encoding small ORFs (smORFs) in primary brown, white, and beige mouse adipocytes. Of these, we validated 85 MPs by proteomics, including 33 circulating MPs in mouse plasma. Analyses of MP-encoding mRNAs under different physiological conditions (high-fat diet) revealed that numerous MPs are regulated in adipose tissue in vivo and are co-expressed with established metabolic genes. Furthermore, Ribo-seq provided evidence for the translation of Gm8773, which encodes a secreted MP that is homologous to human and chicken FAM237B. Gm8773 is highly expressed in the arcuate nucleus of the hypothalamus, and intracerebroventricular administration of recombinant mFAM237B showed orexigenic activity in obese mice. Together, these data highlight the value of this adipocyte MP database in identifying MPs with roles in fundamental metabolic and physiological processes such as feeding.

Comparison of software packages for detecting unannotated translated small open reading frames by Ribo-seq.

Accurate and comprehensive annotation of microprotein-coding small open reading frames (smORFs) is critical to our understanding of normal physiology and disease. Empirical identification of translated smORFs is carried out primarily using ribosome profiling (Ribo-seq). While effective, published Ribo-seq datasets can vary drastically in quality and different analysis tools are frequently employed. Here, we examine the impact of these factors on identifying translated smORFs. We compared five commonly used software tools that assess ORF translation from Ribo-seq (RibORFv0.1, RibORFv1.0, RiboCode, ORFquant, and Ribo-TISH), and found surprisingly low agreement across all tools. Only ~2% of smORFs were called translated by all five tools and ~15% by three or more tools when assessing the same high-resolution Ribo-seq dataset. For larger annotated genes, the same analysis showed ~72% agreement across all five tools. We also found that some tools are strongly biased against low-resolution Ribo-seq data, while others are more tolerant. Analyzing Ribo-seq coverage as a proxy for translation levels revealed that highly translated smORFs are more likely to be detected by more than one tool. Together these results support employing multiple tools to identify the most confident microprotein-coding smORFs, and choosing the tools based on the quality of the dataset and planned downstream characterization experiments of predicted smORFs.

A short ORF-encoded transcriptional regulator.

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.

Repression of the transcriptional activity of ERRα with sequence-specific DNA-binding polyamides.

The orphan nuclear receptors estrogen-related receptors (ERRs) bind to the estrogen-related receptor response element (ERRE) to regulate transcriptional programs in cellular metabolism and cancer cell growth. In this study, we evaluated the potential for a pyrrole-imidazole polyamide to block ERRα binding to ERREs to inhibit gene expression. We demonstrated that the ERRE-targeted polyamide 1 blocked the binding of ERRα to the consensus ERRE and reduced the transcriptional activity of ERRα in cell culture. We further showed that inhibiting ERRα transcriptional activity with polyamide 1 led to reduced mitochondrial oxygen consumption, a primary biological effect regulated by ERRα. Finally, our data demonstrated that polyamide 1 is an inhibitor for cancer cell growth.

Accurate annotation of human protein-coding small open reading frames.

Functional protein-coding small open reading frames (smORFs) are emerging as an important class of genes. However, the number of translated smORFs in the human genome is unclear because proteogenomic methods are not sensitive enough, and, as we show, Ribo-seq strategies require additional measures to ensure comprehensive and accurate smORF annotation. Here, we integrate de novo transcriptome assembly and Ribo-seq into an improved workflow that overcomes obstacles with previous methods, to more confidently annotate thousands of smORFs. Evolutionary conservation analyses suggest that hundreds of smORF-encoded microproteins are likely functional. Additionally, many smORFs are regulated during fundamental biological processes, such as cell stress. Peptides derived from smORFs are also detectable on human leukocyte antigen complexes, revealing smORFs as a source of antigens. Thus, by including additional validation into our smORF annotation workflow, we accurately identify thousands of unannotated translated smORFs that will provide a rich pool of unexplored, functional human genes.

Regulation of the ER stress response by a mitochondrial microprotein.

Cellular homeostasis relies on having dedicated and coordinated responses to a variety of stresses. The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a common stress that triggers a conserved pathway called the unfolded protein response (UPR) that mitigates damage, and dysregulation of UPR underlies several debilitating diseases. Here, we discover that a previously uncharacterized 54-amino acid microprotein PIGBOS regulates UPR. PIGBOS localizes to the mitochondrial outer membrane where it interacts with the ER protein CLCC1 at ER-mitochondria contact sites. Functional studies reveal that the loss of PIGBOS leads to heightened UPR and increased cell death. The characterization of PIGBOS reveals an undiscovered role for a mitochondrial protein, in this case a microprotein, in the regulation of UPR originating in the ER. This study demonstrates microproteins to be an unappreciated class of genes that are critical for inter-organelle communication, homeostasis, and cell survival.

CDK12 phosphorylates 4E-BP1 to enable mTORC1-dependent translation and mitotic genome stability.

The RNA polymerase II (RNAPII) C-terminal domain kinase, CDK12, regulates genome stability, expression of DNA repair genes, and cancer cell resistance to chemotherapy and immunotherapy. In addition to its role in mRNA biosynthesis of DNA repair genes, we show here that CDK12 phosphorylates the mRNA 5' cap-binding repressor, 4E-BP1, to promote translation of mTORC1-dependent mRNAs. In particular, we found that phosphorylation of 4E-BP1 by mTORC1 (T37 and T46) facilitates subsequent CDK12 phosphorylation at two Ser-Pro sites (S65 and T70) that control the exchange of 4E-BP1 with eIF4G at the 5' cap of CHK1 and other target mRNAs. RNA immunoprecipitation coupled with deep sequencing (RIP-seq) revealed that CDK12 regulates release of 4E-BP1, and binding of eIF4G, to many mTORC1 target mRNAs, including those needed for MYC transformation. Genome-wide ribosome profiling (Ribo-seq) further identified specific CDK12 "translation-only" target mRNAs, including many mTORC1 target mRNAs as well as many subunits of mitotic and centromere/centrosome complexes. Accordingly, confocal imaging analyses revealed severe chromosome misalignment, bridging, and segregation defects in cells deprived of CDK12 or CCNK. We conclude that the nuclear RNAPII-CTD kinase CDK12 cooperates with mTORC1, and controls a specialized translation network that is essential for mitotic chromosome stability.

MIEF1 Microprotein Regulates Mitochondrial Translation.

Recent technological advances led to the discovery of hundreds to thousands of peptides and small proteins (microproteins) encoded by small open reading frames (smORFs). Characterization of new microproteins demonstrates their role in fundamental biological processes and highlights the value in discovering and characterizing more microproteins. The elucidation of microprotein-protein interactions (MPIs) is useful for determining the biochemical and cellular roles of microproteins. In this study, we characterize the protein interaction partners of mitochondrial elongation factor 1 microprotein (MIEF1-MP) using a proximity labeling strategy that relies on APEX2. MIEF1-MP localizes to the mitochondrial matrix where it interacts with the mitochondrial ribosome (mitoribosome). Functional studies demonstrate that MIEF1-MP regulates mitochondrial translation via its binding to the mitoribosome. Loss of MIEF1-MP decreases the mitochondrial translation rate, while an elevated level of MIEF1-MP increases the translation rate. The identification of MIEF1-MP reveals a new gene involved in this process.

Tumor Repression of VCaP Xenografts by a Pyrrole-Imidazole Polyamide.

Pyrrole-imidazole (Py-Im) polyamides are high affinity DNA-binding small molecules that can inhibit protein-DNA interactions. In VCaP cells, a human prostate cancer cell line overexpressing both AR and the TMPRSS2-ERG gene fusion, an androgen response element (ARE)-targeted Py-Im polyamide significantly downregulates AR driven gene expression. Polyamide exposure to VCaP cells reduced proliferation without causing DNA damage. Py-Im polyamide treatment also reduced tumor growth in a VCaP mouse xenograft model. In addition to the effects on AR regulated transcription, RNA-seq analysis revealed inhibition of topoisomerase-DNA binding as a potential mechanism that contributes to the antitumor effects of polyamides in cell culture and in xenografts. These studies support the therapeutic potential of Py-Im polyamides to target multiple aspects of transcriptional regulation in prostate cancers without genotoxic stress.

Replication stress by Py-Im polyamides induces a non-canonical ATR-dependent checkpoint response.

Pyrrole-imidazole polyamides targeted to the androgen response element were cytotoxic in multiple cell lines, independent of intact androgen receptor signaling. Polyamide treatment induced accumulation of S-phase cells and of PCNA replication/repair foci. Activation of a cell cycle checkpoint response was evidenced by autophosphorylation of ATR, the S-phase checkpoint kinase, and by recruitment of ATR and the ATR activators RPA, 9-1-1, and Rad17 to chromatin. Surprisingly, ATR activation was accompanied by only a slight increase in single-stranded DNA, and the ATR targets RPA2 and Chk1, a cell cycle checkpoint kinase, were not phosphorylated. However, ATR activation resulted in phosphorylation of the replicative helicase subunit MCM2, an ATR effector. Polyamide treatment also induced accumulation of monoubiquitinated FANCD2, which is recruited to stalled replication forks and interacts transiently with phospho-MCM2. This suggests that polyamides induce replication stress that ATR can counteract independently of Chk1 and that the FA/BRCA pathway may also be involved in the response to polyamides. In biochemical assays, polyamides inhibit DNA helicases, providing a plausible mechanism for S-phase inhibition.