ADARB1 Is Involved In a Reduced Maturation Of The miR15a/Mir-16-1 Family In Chronic Lymphocytic Leukemia
Authors: Mertens Daniel , Allegra Danilo , Garding Angela , Döhner Hartmut , Kuchenbauer Florian , Stilgenbauer Stephan 
Published: October 2019

Abstract
MiRNAs are transcribed as long primary precursors (pri-miRNAs), which are cleaved in the nucleus by DROSHA in combination with cofactors. The product of pri-miRNA cleavage is the precursor miRNA (pre-miRNA), which is exported to the cytoplasm and further cleaved by DICER1 into a functioning miRNA:miRNA* complex.
The MIR15A/-16-1 tumor suppressor microRNAs (miRNAs) are localized in chromosomal band 13q14.3 that is recurrently deleted in more than 50% of patients with chronic lymphocytic leukemia (CLL). MiR-15a/16 are also less abundant in patients without genomic deletion, suggesting that their down regulation in CLL can also be caused by posttranscriptional mechanisms. Intriguingly, in human CLL cells the primary transcripts (pri-miRNAs) of miR-15a/-16/-15b are elevated and processing intermediates (pre-miRNAs) are reduced in cells from CLL patients (n=38) compared to non-malignant b-cells (n=14). 22/38 (58%) of CLL patients displayed a high pri-miR to pre-miR ratio (higher than 1SD of non-malignant cells) and were classified to harbor a putative processing defect. We recently reported epigenetic aberrations at 13q14 in 58/61 (95%) patients, suggesting 13q14 to be affected in all patients. In line with this concept defective processing of 13q14 miRNAs did not correlate with overall survival, cytogenetic aberrations, immunoglobulin mutation status or time since diagnosis.
Using miR-15a/-16-1 pri-miR hairpin sequences coupled to luciferase reporters as DROSHA substrates, intracellular pri-miRNA processing could be shown to be reduced in viable primary CLL cells from patients with a processing defect compared to CLL cells from patients without processing defect and compared to non-malignant B-cells (n=5, n=3, n=4 respectively, p<0.05). This was not seen for processing of pri-miR-155 that was used as negative control. MiRNAs that are putatively affected by the processing defect in addition to the miR-15/-16 family were identified by comparison of the mature miRNome of patients with the processing defect compared to patients without (n=23). In addition to the miR-15/-16 family, in the processing defective patient cells levels of miR-886-3p, miR-7, miR-181d and miR-338-3p were significantly reduced, suggesting that these miRNAs could as well be affected by the processing defect. Intriguingly, the gene targets of these miRNAs are significantly enriched for being downstream members of the immunoglobulin receptor pathway that plays a central role in CLL pathology (KEGG pathways p=2.39 x 10^-9, “miRSystem pathways” p=8 x 10^-4).
In order to characterize the molecular mechanism of the processing defect, the expression of cofactors required for DROSHA processing was quantified with Q-RT-PCR in CLL patients with and without a processing defect (n=38). DROSHA, DGCR8, p68, p72 and KHSRP were not significantly down regulated in CLL cells with a processing defect compared to cells with intact processing. For the identification of candidate genes involved in the processing defect, CLL cell lines were screened for processing activity of pri-miRs. Compared to LCL-WEI, Granta, EHEB, JVM-1, JVM-3 and MEC-1 cells, a reduced processing of pri-miR-16-1 was observed in MEC-2 and I83-E95 cells while pri-miR-34a and pri-miR-155 processing was unaffected. ADARB1 gene was significantly up regulated in the processing defective MEC-2 but not MEC-1 cells. ADARB1 belongs to the family of adenosine deaminases that posttranscriptionally introduce inosines into RNA, and its ectopic overexpression reduced processing of pri-miR-16-1 in cell lines. Intriguingly, this inhibition of processing was not reduced upon removal of the enzymatic deaminating activity of ADARB1, but was abrogated by deletion of the RNA-binding domain and the nuclear targeting of the protein as expected.
Thus, we show that apart from genomic deletion or transcriptional down regulation, aberrant processing of microRNA leads to specific reduction microRNAs in leukemic cells. This represents a novel oncogenic mechanism in the pathogenesis of CLL.

Disclosures:
No relevant conflicts of interest to declare.