Specific U6 Small Nucleolar RNAs Are Present on the Surface of Primary AML Specimens and Are Accompanied By Short Fragments of Longer RNAs

RNA constitutes multiple sub-species with diverse functions and localizations. RNA molecules appear on cellular surfaces although with largely unknown functions. Acute myeloid leukemia (AML) is a particularly aggressive age-related malignancy with a dismal prognosis. We now provide insight into new classes of glycosylated RNAs present on the surface of acute myeloid leukemia (AML) cells from patient samples that may affect AML pathogenesis. We used glyco-cell surface capture technology to specifically label and subsequently enrich surface glycoRNA (surfRNA). We constructed small RNA libraries from surfRNA of activated human T cells, peripheral blasts from AML patient samples and AML cell lines. We performed deep single-end sequencing and compared mapped reads from isolated surfRNA to the corresponding cellular total RNA.

First, we examined the classes of small RNAs and reveal a significant enrichment of small nucleolar RNAs (snRNAs) on the surface of patient AML samples. Specifically, we identify catalytic 100-110nt U6 snRNAs that are important for protein translation, cell proliferation by controlling both the start and end of pre-mRNA splicing (Wang et al. 2023): RNU6-1203P (overlapping SENP1, padj 0.004), RNU6-913P (overlapping C5orf64), RNU6-207P (overlapping IL1RAPL2), RNU6-1316P (overlapping TRAF3). A genetic association between SUMO-specific protease 1 (SENP1) and AML has been validated (Xing et al. 2024). The enrichment of these U6 snRNAs on the AML surface was higher than that on the surface of healthy donor T cells (log2fc > 4; padj < 0.05). We did not find a similarly strong enrichment of other small RNAs including snoRNAs.

RNAs may exist on the surface as ribonucleoprotein (RNP) complexes. Therefore, we inspected our cell surface proteome from matched AML patient samples for U6 snRNA binding proteins. We identified the previously reported SNRNP200 to be highly expressed in our surface proteome, together with other snRNPs PRP4, PRP31, USP39 and pre-mRNA splicing factor proteins PRPF6 and PRPF8. This suggested an expanded class of spliceosome complex proteins on the surface of primary AML. Our gene ontology analysis suggested protein-binding by surfRNA.

Unexpectedly, many longer mRNA, rRNA and lncRNAs were mapped and enriched on the surface of cells as well. Recent studies revealed the existence of very small 18S and 28S rRNA fragments (rRFs). By mapping 25nt rRNA fragments to the 18S and 28S rRNA subunit, we identify rRF sequences commonly enriched on the surface of all cell types (18S_416-519, 18S_1820-1870, 28S_3676-3909, 28S_4351-4533, 28S_1492-1569, 28S_1986-2063) and few regions unique to primary AML samples (18S_1170-1221, 28S_1726-1803, 28S_3338-3363). Common surface rRNA sequences all lie within the static regions of 18S and 28S rRNA. Multiple 10-20 nt reads mapping to lncRNAs PVT-1 (log2fc 6.3, padj < 0.001), BLACAT1 (log2fc 6.4, padj < 0.001) and MIR124-2HG (log2fc 7.6, padj < 0.001) were significantly enriched in surfRNA. Their known interactions with mRNA transcripts of oncogenic genes MYC, CREB1 and BCN1 makes them interesting to further explore in this AML context. To inspect whether our other longer surfRNA indeed comprise full-length reads or fragments, we next performed paired-end sequencing after DNA size exclusion to precisely determine read lengths. With the additional finding of several processed transcripts enriched on the cell surface, we propose that RNA fragments might arise from intracellular RNA processing.

Taken together the spliceosomal U6 snRNA is specifically expressed on primary AML specimens. Its association with corresponding protein partner genes suggests specific function in myeloid leukemia. Further, we report an expanded pool of surfRNA that may in fact only be small RNAs, by size, arising from intracellular RNA processing. Such surfRNAs might serve as novel prognostic biomarkers or targets for AML therapies similar to their well-known partners, surface glycoproteins.