Abstract
Small RNAs have important functions. However, small RNAs in primate oocytes remain unexplored. Herein, we develop CAS-seq, a single-cell small RNA sequencing method, and profile the small RNAs in human oocytes and embryos. We discover a class of ~20-nt small RNAs that are predominantly expressed in human and monkey oocytes, but not in mouse oocytes. They are specifically associated with HIWI3 (PIWIL3), whereas significantly shorter than the commonly known PIWI-interacting RNAs (piRNAs), designated as oocyte short piRNAs (os-piRNAs). Notably, the os-piRNAs in human oocytes lack 2’-O-methylation at the 3’ end, a hallmark of the classic piRNAs. In addition, the os-piRNAs have a strong 1U/10 A bias and are enriched on the antisense strands of recently evolved transposable elements (TEs), indicating the potential function of silencing TEs by cleavage. Therefore, our study has identified an oocyte-specific piRNA family with distinct features and provides valuable resources for studying small RNAs in primate oocytes.
Highlights
The efficient ligation of adapters to scarce small RNAs requires a high concentration of 5′ and 3′ adapters. This requirement produces a high level of adapter heterodimer by-products, which hinder the subsequent amplification of the small RNA complementary DNA libraries[28]
We found that in the presence of the complementary DNA (cDNA) strand generated by reverse transcription (RT), spCas[9] can cleave the RNA–DNA/cDNA chimeras bearing a protospacer adjacent motif (PAM) sequence (TGG) in the 3′ adapter sequence with comparable efficiency to its double-stranded DNA (dsDNA) substrates (Fig. 1b, c, Supplementary Fig. 1a)
To avoid extracting total RNAs from a single cell, which is technically challenging and usually causes a significant loss of RNA content, we used heat to lyse the cell and to release the small RNAs from RNA–protein complexes before ligation with a 3′ adapter. We optimized this procedure by conducting multiple enzymatic reactions on beads. With all of these efforts, we developed CAS-seq (Cas9-assisted small RNA-sequencing) and were able to reduce the input of total RNA to 1 ng or less
Summary
PiRNAs are germ cell-specific small RNAs that form clusters at hundreds of genome locations and are functional in protecting the animal’s germline by silencing transposons and regulating gene expression[7,12,13,14]. PIWI proteins, in complex with primary piRNAs, cleave target RNAs between positions 10 and 11 of the complementary sequence Such cleavage destroys the transposon RNAs and produces the sense-strand piRNA intermediates that bind to the PIWI proteins. Knockout of any of the three Piwi genes in mice causes sterility exclusively in males[25] These species-dependent differences in the impact of PIWI loss raise the question of whether piRNAs have important functions in mammalian female germ cells. We describe a highly sensitive single-cell small RNA-sequencing (RNA-seq) method suitable for detecting low-copy small RNAs and utilize this method to profile small RNAs in human oocytes and early embryos
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