Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide-encoding genes in vertebrates, providing an entry point to define their function in vivo.

Summary Upon fertilization, maternal factors direct development and trigger zygotic genome activation (ZGA) at the maternal-to-zygotic transition (MZT). In zebrafish, ZGA is required for gastrulation and clearance of maternal mRNAs, which is in part regulated by the conserved microRNA miR-430. However, the factors that activate the zygotic program in vertebrates are unknown. Here, we show that Nanog, Pou5f1 and SoxB1 regulate zygotic gene activation in zebrafish. We identified several hundred genes directly activated by maternal factors, constituting the first wave of zygotic transcription. Ribosome profiling revealed that nanog, sox19b and pou5f1 are the most highly translated transcription factors pre-MZT. Combined loss of these factors resulted in developmental arrest prior to gastrulation and a failure to activate >75% of zygotic genes, including miR-430. Our results demonstrate that maternal Nanog, Pou5f1 and SoxB1 are required to initiate the zygotic developmental program and induce clearance of the maternal program by activating miR-430 expression.

Highlights d S. epidermidis strains within-individual are diverse and evolved from multiple founders d Strain diversity is shaped by purifying selection and transmission events d Strain admixture can suppress virulence and alter metabolism at a population level d Horizontal gene transfer disseminates antibiotic resistance genes within individuals

The endonucleolytic cleavage and polyadenylation of a pre-mRNA in mammalian cells requires two c/s-acting elements, a highly conserved AAUAAA hexamer and an amorphous U-or GU-rich downstream element, that together constitute the "core" poly(A) site. The terminal redundancy of the HIV-1 pre-mRNA requires that the processing machinery disregard a core poly(A) site at the 5' end of the transcript, and efficiently utilize an identical signal that resides near the 3' end. Efficient processing at the 3' core poly(A) site, both in vivo and in vitro, has been shown to require sequences 76 nucleotides upstream of the AAUAAA hexamer. In this report we demonstrate that this HIV-1 upstream element interacts directly with the 160-kD subunit of CPSF (cleavage polyadenylation specificity factor), the factor responsible for the recognition of the AAUAAA hexamer. The presence of the upstream element in the context of the AAUAAA hexamer directs the stable binding of CPSF to the pre-mRNA and enhances the efficiency of poly(A) addition in reactions reconstituted with purified CPSF and recombinant poly(A) polymerase. Our results indicate that the dependence of HIV-1 3' processing on upstream sequences is a consequence of the suboptimal sequence context of the AAUAAA hexamer. We suggest that poly(A) site definition involves the recognition of multiple heterogeneous sequence elements in the context of the AAUAAA hexamer.

The full repertoire of human microRNAs (miRNAs) that could distinguish common (benign) nevi from cutaneous (malignant) melanomas remains to be established. In an effort to gain further insight into the role of miRNAs in melanoma, we applied Illumina next-generation sequencing (NGS) platform to carry out an in-depth analysis of miRNA transcriptome in biopsies of nevi, thick primary (>4.0 mm) and metastatic melanomas with matched normal skin in parallel to melanocytes and melanoma cell lines (both primary and metastatic) (n = 28). From this data representing 698 known miRNAs, we defined a set of top-40 list, which properly classified normal from cancer; also confirming 23 (58%) previously discovered miRNAs while introducing an additional 17 (42%) known and top-15 putative novel candidate miRNAs deregulated during melanoma progression. Surprisingly, the miRNA signature distinguishing specimens of melanoma from nevus was significantly different than that of melanoma cell lines from melanocytes. Among the top list, miR-203, miR-204-5p, miR-205-5p, miR-211-5p, miR-23b-3p, miR-26a-5p and miR-26b-5p were decreased in melanomas vs. nevi. In a validation cohort (n = 101), we verified the NGS results by qRT-PCR and showed that receiver-operating characteristic curves for miR-211-5p expression accurately discriminated invasive melanoma (AUC = 0.933), melanoma in situ (AUC = 0.933) and dysplastic (atypical) nevi (AUC = 0.951) from common nevi. Target prediction analysis of co-transcribed miRNAs showed a cooperative regulation of key elements in the MAPK signaling pathway. Furthermore, we found extensive sequence variations (isomiRs) and other non-coding small RNAs revealing a complex melanoma transcriptome. Deep-sequencing small RNAs directly from clinically defined specimens provides a robust strategy to improve melanoma diagnostics.