CircRNAs in hematopoiesis and hematological malignancies

Abstract

Cell states in hematopoiesis are controlled by master regulators and by complex circuits of a growing family of RNA species impacting cell phenotype maintenance and plasticity. Circular RNAs (circRNAs) are rapidly gaining the status of particularly stable transcriptome members with distinctive qualities. RNA-seq identified thousands of circRNAs with developmental stage- and tissue-specific expression corroborating earlier suggestions that circular isoforms are a natural feature of the cell expression program. CircRNAs are abundantly expressed also in the hematopoietic compartment. There are a number of studies on circRNAs in blood cells, a specific overview is however lacking. In this review we first present current insight in circRNA biogenesis discussing the relevance for hematopoiesis of the highly interleaved processes of splicing and circRNA biogenesis. Regarding molecular functions circRNAs modulate host gene expression, but also compete for binding of microRNAs, RNA-binding proteins or translation initiation and participate in regulatory circuits. We examine circRNA expression in the hematopoietic compartment and in hematologic malignancies and review the recent breakthrough study that identified pathogenic circRNAs derived from leukemia fusion genes. CircRNA high and regulated expression in blood cell types indicate that further studies are warranted to inform the position of these regulators in normal and malignant hematopoiesis.

Figure 1

Linear and circRNAs. CircRNAs are produced by backsplicing, and combinations of exons and introns give rise to different products, including single circularized exons, circRNAs formed by two or more exons, by exon and retained intron sequences (EI-ciRNAs) and by intronic sequences only.

Figure 2

Molecular methods for circRNA detection, validation and study. (a) CircRNA detection from RNA-seq data grounds on the identification of sequence reads encompassing the backsplice junction; (b) Backsplice reads map to the genome in chiastic order (two segments of a single read align separately in reverse order) due to the backsplicing in circRNAs biogenesis. (c) Convergent primers (white arrows) designed in adjacent spliced exons amplify both linear and circular isoforms, whereas primers that are divergent in the linear transcripts (black arrows) can be used to specifically amplify the circular isoform; (d) PolyA enrichment protocols deplete circRNAs, whereas ribosome depletion and RNAse R protocols enrich circRNAs; (e) RNAse R digestion before reverse transcription–PCR lowers the amount of false-positive amplicons facilitating circRNA validation; (f) Gel Trap electrophoresis allows isolate the circular and linear fractions of the input RNA, as circRNAs are hold in the well; (g) Two-dimensional acrylamide gel electrophoresis separates the circular RNA fraction in an off-diagonal curve; (h) RNA migration in agarose gel before and after a mild RNAse H treatment resulting in a single cut per molecule shows that circular molecules bearing a ‘backsplice' junction are discriminated from linear ones deriving from a duplication event, as only circRNA results in a single band after being cut once (f–h re-elaborated from).

Figure 3

Expression variation of enzymes involved in circRNA expression. Gene expression intensities of ADAR1, MBNL1 and QKI in samples of normal bone marrow and six B-cell leukemia subtypes carrying specific genetic aberrations (according to Haferlach et al.); expression data obtained with HG-U133 Plus 2.0 (Affymetrix, Santa Clara, CA, USA).

Figure 4

CircRNA functions. Elucidated circRNA functions include the ability to sponge miRNAs thus regulating the silencing of canonical targets (for example, ciRS-7/CDR1-as harbors 63 binding sites for miR-7) and participating to ceRNA networks; similarly circRNAs could decoy RBP ultimately regulating the functions in which RBP are implicated (for example, circ-Foxo3 forms a ternary complex with p21 and CDK2 arresting cell cycle progression); circRNAs can also regulate in cis the expression if the gene from which they derive through interactions with the U1 RNA in the U1 RNP in the nucleus (for example, circEIF3J); moreover, circRNAs harboring an IRES could be translated to produce peptides or compete with mRNA translation (for example, circFMN contains an active translation start site not leading to the protein synthesis).

Figure 5

f-circRNAs derived from chromosomal translocations have oncogenic role. (a) Transcription of fusion genes generated by cancer-associated chromosomal translocation could generate both linear mRNA coding for oncogenic fusion proteins and f-circRNAs. The figure depicts the example of f-CircM9_1 expressed in cells harboring the well-known acute myeloid leukemia MLL/AF9 t(9;11)) translocation: f-CircM9_1 includes two sequences not present in the normal genome, the MLL exon 8 and AF9 exon 6 fusion junction derived from the chromosomal translocation, and the backsplice junction connecting MLL exon 7 with AF9 exon 6; (b) f-CircM9 was demonstrated to be proto-oncogenic in vitro (increasing proliferation rate and foci forming ability in mouse embryonic fibroblasts, MEF), and required for leukemic cell (THP1) viability. f-CircM9 alone resulted not sufficient to trigger leukemia in vivo when expressed in HSC xenografted in mice. Concurrent expression of f-circM9 and MLL/AF9 fusion protein contributed to leukemia progression in vivo and ex vivo cells expressing f-circM9 and MLL/AF9 displayed increased ability to proliferate and to form colonies. Furthermore, f-circM9 expression in MLL/AF9 mouse model cells increased the resistance to leukemia treatments suggesting that f-circM9 impacts to pre-clinical therapeutic outcome.