Results

Although there are many suggestions that the circularization of RNAs arise by back-splicing, it does not fully explain its specificity or selectivity (Kristensen et al., 2019; Pervouchine, 2019). To get a better insight into the mechanism of the process, first we modeled the secondary structure of antisense and sense CDR1 transcripts (Figure S1). As one can see, the predicted structure of both RNAs shows many double-stranded stable fragments at which microRNA binding sites (pentanucleotide CUUCC) are identified. In the most sites, they are hidden and are not available to pair with the miR-7 or miR-671 seed sequence. In such cases, sponging capacity, the most important function of circRNAs is excluded. Free energy for predicted secondary structure RNA shows ΔG −339.50 and −161.81 kcal/mol for CDR1 RNA and CDR1as RNA, respectively. Although the calculated CDR1as RNA structure is less stable than its sense counterpart, it is clear that direct binding miR-7 or miR-671 to circular CDR1as RNA is rather difficult or even impossible.

A new molecular mechanism of RNA circularization and the microRNA sponge formation

All authors

Agnieszka Belter , Mariusz Popenda , Marcin Sajek , Tomasz Woźniak , Mirosława Z. Naskręt-Barciszewska , Marta Szachniuk , Stefan Jurga & Jan Barciszewski

https://doi.org/10.1080/07391102.2020.1844802

Published online:

17 November 2020

Figure 1. Primary structure of CDR1as RNA and miR-7. (a) Nucleotide sequence of CDR1as RNA (1299 nt) with marked 71 CUUCC sequences (yellow). Pentapyrimidine CUUCC sequence appears regularly from 26th to 70th CUUCC sequences and are variable at the 5’ end of CDR1as (nucleotides 1–480). (b) Nucleotide sequence of miR-7 (in blue) with GGAAG pentanucleotide ‘seed region’(underlined).

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Looking for a precise mechanism of circular RNA formation, we carried out the in-deep nucleotide analysis of CDR1as RNA. We found 71 pentanucleotide CUUCC sequences (seed binding sites, SBS), which are distributed regularly throughout the entire molecule (Figure 1, Figures S2a,b). The multiple alignments and the nucleotide occupancy analysis of CDR1as RNA showed a very high SBS sequence conservation at all RNA positions (Figure S2b,c).

A new molecular mechanism of RNA circularization and the microRNA sponge formation

All authors

Agnieszka Belter , Mariusz Popenda , Marcin Sajek , Tomasz Woźniak , Mirosława Z. Naskręt-Barciszewska , Marta Szachniuk , Stefan Jurga & Jan Barciszewski

https://doi.org/10.1080/07391102.2020.1844802

Published online:

17 November 2020

Figure 2. The structure of the complex of CDR1as RNA with miR-7 and miR-671. (a) The binding of three miR-7s (n-1, n, n + 1) to CDR1as RNA shows three structural domains of the complex: (i) two short helixes (helixes 1 and 2) separated with internal loop, (ii) the hinge and (iii) the triple-helix. (b) The binding of miR-671 to CDR1as RNA does not show the hinge and the triple helix. (c) The detailed secondary structure of the complex of CDR1as RNA with three consecutive miR-7s. In addition to canonical Watson-Crick pairs, G = C and A-U pairs, there are non-standard base pairs U-•-U, U-•-C and A-•-A in the bulge and hinge. The Hoogsteen base pairs in the triplex are marked as -■-. (d) The tertiary structure of the fragment of the complex of CDR1as RNA with two consecutive miR-7s. (e) Ring-like CDR1as RNA complexed with 71 miR-7s. (a–e) CDR1as – black, miR-7 – blue, miR-671 – green, GGAAG – seed sequence of miR-7, CUUCC – yellow – the sequence of CDR1as RNA perfectly complementary to seed regions of miR-7 and miR-671.

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Next, the sequence of miRNAs were analyzed (Kozomara & Griffiths-Jones, 2014). We found five miRNAs, particularly miR-7, miR-671, miR-3202, miR-4533, and miR-6828, out of 2813 currently known miRNAs (miRBase), that contain the conserved GGAAG seed sequence, at positions 2–6 of their 5’ end (Figure S3a) (Ambros, 2004; Bartel, 2004). The analysis of a putative secondary structure of these five miRNAs showed that only miR-7 does not show the propensity to form a secondary structure, but the others are prone to form a hairpin structure (Figure S3b). That observation suggests that CDR1as RNA, through its SBS, can form the short double helix with miR-7. Because of the identified seed sequence, similar RNA-RNA interactions with miR-671 are also possible.

A new molecular mechanism of RNA circularization and the microRNA sponge formation

All authors

Agnieszka Belter , Mariusz Popenda , Marcin Sajek , Tomasz Woźniak , Mirosława Z. Naskręt-Barciszewska , Marta Szachniuk , Stefan Jurga & Jan Barciszewski

https://doi.org/10.1080/07391102.2020.1844802

Published online:

17 November 2020

Figure 3. The mechanism of miR-7 induced ligation of CDR1as RNA. (a) Binding of the 1st, 2nd, and 71st miR-7s to CDR1as RNA. The 1st miR-7 pairs with CDR1as RNA, and gets together of the 3’ end (nucleotide A₁₂₉₉) and the 5’ end (nucleotide C₁) of CDR1as RNA and promotes their ligation. The triple helix is not found. (b) miR-671 does not show the capacity to get together of both ends of CDR1as RNA. (c) The structure of the CDR1as RNA ligation site triggered by miR-7 (1st), which directs the covalent binding of A₁₂₉₉ with C₁. (d) The tertiary structure of the complex of CDR1as RNA and miR-7 showing the ligation site. (a–d) CDR1as – black, miR-7 – blue, miR-671 – green, GGAAG – seed sequence of miR-7, CUUCC – yellow – the sequence of CDR1as RNA perfectly complementary to seed regions of miR-7 and miR-671. For details see also Figures S4–S6.

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An in-deep analysis of the distribution of seed sequence suggests that along CDR1as RNA circularization, sequential binding of miR-7s is favorably possible. That newly synthesized unstructured fragments of transcribed RNA are good targets for miR-7 RNA binding, much better than full-length transcript with a strong secondary structure. One should remember that both miR-7 and CDR1as RNA co-express to high levels in neuronal tissues, especially the cerebellum and midbrain (Choudhury et al., 2013; Kleaveland et al., 2018; Piwecka et al., 2017) and both RNAs colocalize in the nucleus (Castanotto et al., 2009, 2018; Liao et al., 2010). Furthermore, miR-7s bind to CDR1as RNA and form a polypurine-polypyrimidine duplex. The double stranded parts facilitate bending of the miR-7/CDR1as double helix, which is proceed as long as the last and the first nucleotides, at the 3′ and 5’end of CDR1as RNA, respectively, come to close proximity. Such arrangement stimulates phosphodiester bond formation. This model explains the prevalence of circular isoform of CDR1as transcript (Barrett et al., 2017; Hansen et al., 2011, 2013; Memczak et al., 2013). Detailed analysis of miRNA/CDR1as complex, showed that each of 71 fully conserved CUUCC motifs of CDR1as RNA, perfectly matches the GGAAG seed sequence of miR-7 and miR-671. Each short double helix is followed by 12- and 13-nt fragments of CDR1as RNA with a partial complementary to miR-7 and miR-671, respectively (Figures S4–S6). Interestingly, the 3′ end of miR-7 does not show a full complementarity to CDR1as RNA, but nucleotides at positions 18–23 of the 3′ end of miR-671, fully match the adjacent CUUCC seed binding site (Figures S4–S6).

To get more insight into specificity of RNA circularization, we calculated the free energy of the structures of the complexes of miR-7 and miR-671 with different fragments of CDR1as RNA. The calculated minimal free energies (MFEs) of these potential RNA-RNA complexes are shown in Table S1. The MFEs of RNA-RNA hybrids with miR-7 are similar and vary from −8.1 (26) to −22.6 (8) kcal/mol (the discrepancy range of MFEs is 14.5 kcal/mol) (see Table S1). Fragments with miR-671 show MFE in the range of −9.7 (54) to −41.9 (62) kcal/mol (Figure S7). A high discrepancy of MFE of miR-671 hybrids (32.2 kcal/mol) shows that some CDR1as RNA fragments, especially those with CUUCC motifs 67 and 39 of highest MFE, are preferentially paired with miR-671 (Figure S8). The average MFE for hybrids with miR-7 is 13.95 kcal/mol and is significantly lower than for all hybrids with miR-671 (20.6 kcal/mol) (Figures S4–S7). The differences in calculated stability of RNA-RNA complexes are a result of the lack of the complementarity of the 3′ end of miR-7 and of miR-671 with CDR1as RNA. The observed energy and the structure differences show functional consequences. The single miR-7 can form Watson-Crick base pairs with the CUUCC sequence, therefore, 1 molecule CDR1as RNA binds with 71 molecules of miR-7s and forms stable structure with total MFE of 990.9 kcal/mol (stabilising effect of triplex has not been taken into consideration) (Figure S9). On the other hand, the single miR-671 binds two consecutive CUUCC sequences and therefore, its loading capacity to CDR1as RNA is 2x lower. Thus, 36 miR-671 molecules match to CDR1as RNA when the first hybrid includes the 1st and 71st CUUCC sequences, and 35 of miR-671 molecules when 1st and 2nd CUUCC sequences are involved (Figures S4–S6). The total MFEs of these two alternative structures show 759.2 and 704.8 kcal/mol for 1 molecule of CDR1as with 36 molecules of miR671 and 1 molecule of CDR1as with 35 molecules of miR671, respectively (Figure S9).

A detailed analysis of the whole complex of CDR1as RNA with 71 molecules of miR-7s showed three specific structural domains, starting with (i) two short helixes with an internal loop. The first helix (helix 1) contains the seed sequence GGAAG, at positions 2–6 at 5´end of miR-7, which perfectly match the CUUCC sequence. The second helix (helix 2) follows the internal loop. The second domain is the hinge (ii). The triple helix, where the 3′ end of miR-7, binds to helix 1 through Hoogsteen hydrogen bonds, forms the third domain (iii) (Figure 2 and Figure S4). All these domains appear regularly and show sequential, one by one mode of binding of 71 miR-7 molecules to single CDR1as RNA (Figure 2 and Figure S4). That specific binding of the GGAAG seed sequence of miR-7 to complementary CUUCC fragment of CDR1as, triggers RNA-RNA complex formation and its bending, which brings together both ends of CDR1as RNA. The double-stranded RNA curvature has already been observed in the Shine-Dalgarno sequence of the 3′ end of 16S rRNA and the 5′ end of mRNA (Shine & Dalgarno, 1975). It is the necessary but insufficient requirement for RNA circularization. The most important and critical step in circularization is a double-stranded RNA formation of 1st miR-7 with the both ends of CDR1as RNA (Figure 3). So, the 3’ end (nucleotide A₁₂₉₉) and the 5′ end (nucleotide C₁) of CDR1as RNA can be precisely ligated and subsequently form circle-like (ring) structure (Figure 2).

Thus, we showed for the first time that CDR1as RNA circularization depends on precise sequential binding of miR-7, which is stabilized by the triple helix formation. Such mode of miR-7 binding perfectly explains the function of CDR1as RNA as the sponge, where the ligand (miR-7) molecule binds to CDR1as RNA with a ratio of two orders of magnitude higher (71:1). It is not surprising, because in some tissues, both CDR1as RNA and miR-7 occur at a high level (Memczak et al., 2013). If so, it is clear that circularization depends on the availability of miR-7, which facilitates the triple-helix formation with CDR1as RNA. The triplex stabilizes the complex, induces nuclease resistance and excludes RISC formation, making the circular RNA very stable (Memczak et al., 2013). The protective function of RNA triplex with Hoogsteen bonds has already been observed for the higher-order structure at the lncRNA MALAT1 3′ terminus, which protects the transcript from degradation (Ageeli et al., 2018; Brown et al., 2016; Ruszkowska et al., 2018). Recently, it has been found that miRNAs can also form a triple helix with double-stranded purine-rich sequences of gene promoters and modify gene transcription (Paugh et al., 2016). All these observations support that CDR1as RNA functions as the sponge and scavenger of a freely available miR-7, prevents it from interacting with unspecific targets (Piwecka et al., 2017).

One should also notice that the complexes of miR-7 and miR-671 with different fragments of CDR1as RNA show significantly different tertiary structures, although observed Watson-Crick interactions are similar (Figures 2 and 3, Figures S4–S6). miR-671 binding in contrast to miR-7 is not able to bring both CDR1as RNA ends close to each other, which is required for RNA circularization (Figure 3). The binding sites for miR-671 showed almost full complementarity to CDR1as RNA (Figure 2), and therefore, miR-671 can mediate slicing of CDR1as RNA in an Ago2-slicer-dependent manner (Hansen et al., 2011). The formation of the triple helix is impossible, therefore, it becomes clear that upon miR-671 binding, CDR1as depletion will take place once miR-7 is released from the complex (Hansen et al., 2013).

To check the same mechanism in other circRNAs, we analyzed over 341 000 human circRNAs deposited in the recent CircAtlas database that contains almost 1.1 million entries (Wu et al., 2020). We found a huge number circRNAs rich in miRNA seed, binding repeated sequences. For example, there are 1476 and 46 human circRNAs containing at least 20 and 50 pentanucleotide repeats, respectively (Tables S2 and S3). Most of them have more than one set of single repeats. On the other hand, hsa-intergenic_009898, of 1465 nucleotide length, contains AUGGA, AAUGG, GGAAU, and AGGAA motifs repeated 135, 133, 116, and 109 times, respectively. Similar circRNAs, which have at least one pentanucleotide repeated at least 60 times can be found Table S4.

Furthermore, we analyzed repeated sequences within circRNAs and found that most of them occurred regularly. To show that mechanism of RNA circularization concerns not only brain, but also other tissues, we analyzed stomach-specific has-PHF2_0015. One can see AGGUG pentanucleotide motif repeated regularly 60 times within that circRNA (Figure S10a and S11a). Within has-PHF2_0015 RNA, we identified 110 fragments of 12 nucleotide length and two shorter of 10- and 6-nt) fragments of very similar sequence. 60 of them have at least one AGGUG sequence motif at 5′ end of the sequence (Figures S10 and S11). The other fragments have similar sequences in this position, with one substitution as: AGG(C)UG and AGG(U)UG (1 fragment), AGG(A)UG (14 fragments) or two substitutions, i.e. A(G)GGU(A)G and A(G)GG(A)UG (19 and 16 fragments, respectively). When the ends of RNA are in close proximity, 61st AGGUG binding site is created. The consensus sequence of these fragments is AGGUGCAGGUGU. The sequence AGGUG at its 3′ end perfectly matches CACCU seed sequence at positions 2–6 at the 5´end of miR-3622a-3p RNA (Figures S10b and S11b).

Furthermore, in addition to miR-3622a-3p, we found other miRNAs, as miR-1306-5p, miR-3622b-3p, and miR-6765-3p, that have also CACCU seed sequence, being perfectly complementary to AGGUG regularly repeated sequences within has-PHF2_0015 RNA (Figure S12). Although, the sequence similarity between these miRNAs, beyond the seed region is very low, the high similarity (86%) of miR-3622a-3p and miR-3622b-3p sequences is observed. Furthermore, the secondary structure analysis of these miRNAs showed that miR-1306-5p, miR-3622a-3p, miR-3622b-3p have no propensity to form a secondary structure (Figure S12). Only miR-6765-3p is prone to form the hairpin structure. One can notice that also in this case the seed sequence could easily bind AGGUG sequence of has-PHF2_0015 RNA.

To get better insight into the role of these miRNAs in the specificity of RNA circularization, we calculated the free energies of the structures of the complexes of these miRNAs with different fragments of has-PHF2_0015. The calculated minimal free energy (MFE) showed that miR-3622a-3p is the most prompt to bind all fragments of has-PHF2_0015, and what is the most interesting, it gets together of the 3’ end (nucleotide G₁₃₃₂), and the 5′ end (nucleotide G₁) of has-PHF2_0015 RNA could facilitate its ligation (Figure S13)

A detailed analysis of the whole complex of has-PHF2_0015 RNA with miR-3622a-3p showed three specific structural domains similar to these observed for CDR1as RNA and miR-7. They are (i) short helixes separated with internal loop, (ii) the hinge, and (iii) the triple-helix (Figure S13a) All domains appear regularly and show sequential mode of binding of 112 miR-3622a-3p molecules to single has-PHF2_0015 RNA. The specific binding of the CACCU seed sequence of miR-3622a to complementary AGGUG fragment of has-PHF2_0015, triggers RNA-RNA complex formation and its bending, which brings together both ends of has-PHF2_0015 RNA. The most important and critical step in circularization is a double-stranded RNA formation of 1st miR-3622a-3p to the both ends of has-PHF2_0015 RNA (Figure S13b). So, the 3’ end (nucleotide G₁₃₃₂) and the 5′ end (nucleotide G₁) of has-PHF2_0015 RNA can be precisely ligated and subsequently form circle-like (ring) structure.