# Evaluation of microRNA variant maturation prior to genome edition

Isabelle Busseau, Sophie Mockly, Élisabeth Houbron, Hedi Somaï, Hervé Seitz

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## To cite this version:

Isabelle Busseau, Sophie Mockly, Élisabeth Houbron, Hedi Somaï, Hervé Seitz. Evaluation of microRNA variant maturation prior to genome edition. Biochimie, 2024, 217, pp.86-94. 10.1016/j.biochi.2023.06.007. hal-04661180

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Distributed under a Creative Commons Attribution 4.0 International License
# Evaluation of microRNA variant maturation prior to genome edition☆

Isabelle Busseau*, Sophie Mockly<fnref n="1" />, Élisabeth Houbron, Hedi Somaï, Hervé Seitz*

Institut de Génétique Humaine, UMR 9002, CNRS and University of Montpellier, Montpellier, France

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

**Article history:**

Received 16 December 2022

Received in revised form

22 May 2023

Accepted 13 June 2023

Available online 27 June 2023

Handling Editor: Dr B Friguet

For the purpose of Open Access, a CC-BY public copyright licence has been applied by the authors to the present document and will be applied to all subsequent versions up to the Author Accepted Manuscript arising from this submission.

**Keywords:**

microRNA

bantam

Drosha

Genome editing

## ABSTRACT

Assessment of the functionality of individual microRNA/target sites is a crucial issue. Genome editing techniques should theoretically permit a fine functional exploration of such interactions, allowing the mutation of microRNAs or individual binding sites in a complete *in vivo* setting, therefore abrogating or restoring individual interactions on demand. A major limitation to this experimental strategy is the influence of microRNA sequence on its accumulation level, which introduces a confounding effect when assessing phenotypic rescue by compensatorily mutated microRNA and target site. Here we describe a simple assay to identify microRNA variants most likely to accumulate at wild-type levels even though their sequence has been mutated. In this assay, quantification of a reporter construct in cultured cells predicts the efficiency of an early biogenesis step, the Drosha-dependent cleavage of microRNA precursors, which appears to be a major determinant of microRNA accumulation in our variant collection. This system allowed the generation of a mutant *Drosophila* strain expressing a *bantam* microRNA variant at wild-type levels.

© 2023 Published by Elsevier B.V.

## 1. Introduction

microRNAs (miRNAs) are small regulatory RNAs which guide a repressive ribonucleoproteic complex onto specific RNA targets. miRNA targets are recognized by sequence complementarity, with most known targets in animals exhibiting a perfect complementarity to the miRNA “seed” sequence (nt 2–7), and imperfect complementarity to the rest of the miRNA sequence [1]. While computational tools, and high-throughput molecular biology methods, typically predict hundreds of RNA targets for each miRNA, *in vivo* genetics usually identifies just a few key targets responsible for miRNA-controlled phenotypes [2–6].

Such discrepancy between molecular biology and bioinformatics on one hand, and *in vivo* genetics on the other hand, is a central question in miRNA biology. The development of CRISPR/Cas9-mediated genome editing now offers a great opportunity to assess it rigorously, with the possibility to mutate miRNA genes, individual miRNA binding sites on target RNAs, or both of them simultaneously (offering the possibility to restore a single interaction with compensatory mutations in the miRNA and its target) [3]. It can be expected that such precise genetics experiments will disprove the biological functionality of many published miRNA/target interactions, as exemplified by the *bantam/enabled* interaction in *Drosophila* [7,8].

miRNA genes are typically transcribed as long precursors called “pri-miRNAs”. In animals (see Ref. [1] for a review), most pri-miRNAs are cleaved in the nucleus by the Drosha endonuclease, liberating a short (≈70 nt) stem-loop-folded “pre-miRNA”. The pre-miRNA is exported to the cytoplasm, where most pre-miRNAs are cleaved by the Dicer endonuclease: the cleavage reaction liberates the loop (which is quickly degraded) from the stem. The stem is composed of two small RNAs which are essentially complementary to each other. That duplex is then loaded on a protein of

<footnote>* Distributed under a Creative Commons Attribution | 4.0 International licence: CC-BY 4.0.</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>*E-mail addresses:* isabelle.busseau@igh.cnrs.fr (I. Busseau), herve.seitz@igh.cnrs.fr (H. Seitz).</footnote>

<footnote n="1">¹ Current address: Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC, Canada.</footnote>
the Ago subfamily of the Argonaute family, and one strand is ejected and quickly degraded (see Ref. [9] for a review). In general for a given miRNA gene, the same RNA strand tends to remain associated to Ago (that strand is called the “miRNA”), while the other strand tends to be most frequently ejected and degraded (that other strand is called the “miRNA*”).

Known determinants of miRNA maturation from their pri-
mRNA and pre-miRNA precursors rely on sequence and second-
ary structure motifs, none of them being located in the miRNA/
miRNA* duplex [10–12]. But in addition to these well-defined
motifs, miRNA maturation relies on the stability of the stem-loop
structure, and mutations affecting the double-stranded structure
of the stem may also perturb miRNA biogenesis. This dependency
creates a major obstacle to the generation of useful genome-edited
miRNA genes, complicating the interpretation of biological phe-
notypes when a mutated miRNA is not only modified in its
sequence, but also in its expression level [3].

Here we describe the generation of genome-edited alleles of the *bantam* miRNA in *Drosophila*, showing that miRNA seed mutation can profoundly perturb miRNA biogenesis. In that example, a simple cell culture assay can identify miRNA mutants which are inefficiently processed, therefore limiting the risk of preparing mutant animal strains with defective mutant miRNA biogenesis. We applied this method to generate a *Drosophila* strain where the *bantam* seed sequence has been mutated to another hexamer, and where mutant *bantam* accumulates at very similar levels as wild-type *bantam*.

**2. Materials and methods**

Flies were raised under standard culture conditions at 25 °C.

2.1. Molecular biology

Preparative PCR for plasmid construction were performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs cat. #M0491S) following the manufacturer's protocol. All constructs were verified by Sanger sequencing (Eurofins). Their annotated sequences are available at https://github.com/HKeyHKey/Busseau_et_al_2023/tree/main/Sequences.

## 2.2. Genome editing

Donor plasmids were prepared by PCR-amplifying chr3L bp 640650-643906 (dm6 assembly) from genomic DNA from *Drosophila* strain *nos-Cas9* (Bloomington *Drosophila* Stock Center #54591) with primers d1319 and d1320 (see Supplementary Table 1 for oligo sequences), then cloning that product between the *Sacl* and *Xhol* sites of pBTKS-. Mutant constructs were generated by PCR-mediated mutagenesis on that plasmid, then replacing the *Agel/HindIII* fragment by the mutated fragment.

sgRNA-expressing plasmids were prepared by cloning oligo
duplexes in the BbsI site of plasmid pCFD3-dU6:3gRNA, which was
a gift from Simon Bullock (Addgene plasmid #49410; http://n2t.
net/addgene:49410; RRID: Addgene_49410; [13]). sgRNA speci-
ficity was predicted using CRISPR targetFinder (http://targetfinder.
flycrispr.neuro.brown.edu/). For constructs inserted using the PAM
located 9 nt downstream of the hairpin, the cloned duplex was
made by annealing oligos d1316 and d1343; for constructs inserted
using the PAM located 52 nt downstream of the hairpin, the cloned
duplex was made by annealing oligos d1938 and d1939.

Edits were performed following the protocols described by
Refs. [13,14]: donor plasmid (300–700 ng.µL⁻¹) and sgRNA pro-
ducer plasmid (100–150 ng.µL⁻¹) were co-injected into nos-Cas9
embryos (Bloomington *Drosophila* Stock Center strain BL#54591)

using TheBestGene service (https://www.thebestgene.com/) or in
our own facility. G0 adults were then crossed to the balancer
BL#23232 strain (TM6B-YFP/Dr), their progeny was screened by PCR
using oligos specific for the intended mutation, and heterozygous
stocks were established and maintained on the TM6B-YFP balancer
chromosome from BL#54591. All edited bantam alleles were veri-
fied by Sanger sequencing (Eurofins).

2.3. *Small RNA-Seq*

Total RNA was TRIZOL-extracted (Life Technologies) from approximately 100 L1 heterozygous larvae of each genotype, hand-collected under a fluorescent binocular equipped with a GFP filter set. For Fig. 1C, larvae were collected 0–2 h after hatching; for Fig. 4B, they were collected 0–6 h after hatching. Samples were then depleted of 2 S rRNA by Oligo Capture using biotinylated oligo d2227 and magnetic Dynabeads MyOne Streptavidin C1 (Life Technologies cat. #65001) following the protocol described by Ref. [15]. Spike-in RNA oligos were then introduced in the RNA samples: for the experiment shown in Fig. 1, an equimolar mix of oligos r25 and r26 was added to each RNA sample (1 µL of a mix containing 5 × 10⁻¹⁶ M of each oligo, per µg of total RNA). This spike-in mix was too dilute and spike-in counts were not useable for the four libraries. Another library was then prepared using an aliquot of the “m2(proximal)/+” RNA sample supplemented with a more concentrated oligo mix (1 µL of a mix containing 1.5 × 10⁻¹⁰ M of each oligo, per µg of total RNA), and its spike-in counts were used to correct for sequencing biases in the first four libraries.

For the experiment shown in Fig. 4, an equimolar mix of RNA
oligos r25, r26 and r64 was added to each RNA sample (1 µL of a mix
containing 1.5 × 10⁻¹⁰ M of each oligo, per µg of total RNA).

Small RNA-Seq libraries were prepared by the MGX sequencing facility using the NEXTflex Small RNA-Seq Kit v3 (Bioo scientific), with pre-adenylated 3' adapter sequence being NNNNTGGAA TTCTCGGGTGCAAGG. Libraries were quantified on a Fragment Analyzer (Agilent) using kit "High sensitivity NGS" and by qPCR on a Roche LightCycler 480.

3' adapter sequences were trimmed, and reads 18-30 nt long
were selected, using cutadapt. Adapter-trimmed reads were map-
ped by Hisat2 on an index composed of the *Drosophila* dm6
genome, supplemented with sequences of the spiked-in RNA oligos
as well as the sequences of the edited *bantam* hairpins. Full-length
spike-in sequence occurences were counted in the cutadapt-
trimmed fastq files, while wild-type and mutant *bantam* read
counts were extracted from the hisat2 mapping output. Read
counts were normalized to the total number of small RNA reads
mapping on the hisat2 index.

2.4. *Luciferase reporter assays*

Reporter plasmids containing wild-type, md, m1, m2, m3 or m4
*bantam* variants were constructed by cloning the hairpin of interest
(PCR-amplified with oligos d2289 and d2290 from the existing
donor DNA, see above) into the *Sacl* and *Xbal* sites of the pmirGLO
vector (Promega cat. #E1330).

Reporter plasmids containing m5, m6, m7, m8 or m9 variants were constructed using NEBuilder HiFi DNA Assembly (NEB cat. #E2621) by assembling a PCR fragment amplified using oligos d2436 and d2437 from the reporter plasmid pmirGLO containing wild-type *bantam*, together with oligo duplexes made by annealing oligos d2438 and d2439 (m5), d2440 and d2441 (m6), d2442 and d2443 (m7), d2444 and d2445 (m8), or d2446 and d2447 (m9).

S2R+ cells [16] from DGRC were cultivated under standard conditions at 25 °C in Schneider medium with 10% Foetal Bovine
medium per well). After two days they were transfected and an additional 10 µg dsRNA was added per well. Transfections were performed in duplicate into 8 of the wells with pmiR-GLO-banmd, banwt, banm2 and banm6, and two wells were mock-transfected. Eight days after the initial dsRNA treatment, cells transfected with pmiR-GLO derivatives were processed for Luciferase assays, and the mock-transfected cells were washed with PBS then scraped in 1 mL TRIZOL (Thermo Fisher Scientific) for RNA extraction, followed by DNase I treatment (RQ1, Promega) and ethanol precipitation. The quality of RNA was estimated by standard agarose gel electrophoresis and the concentration was determined on a Nanodrop spectrophotometer.

One microgram total RNA was reverse transcribed using oligo-dT 12-18 nt long (Life Technologies) and SuperScript II reverse transcriptase (Life Technologies). Quantitative PCR was performed using the LightCycler 480 SYBR Green I Master system (Roche) with the housekeeping genes *Actin 42A* and *Tubulin 84B* as references. Primers were d2638 and d2639 (*Drosha*), d2642 and d2637 (*Exportin-5*), d1654 and d1655 (*Actin 42A*), and d1658 and d1659 (*Tubulin 84B*).

## 2.6. *In vivo* GFP reporter assays

The GFP reporter plasmid containing a wild-type *bantam* hairpin in its 3' UTR was constructed as follows: a GFP-*bantam* fragment was amplified using oligos d1360 and d1361 from the transgenic fly strain containing UAS-banD [18], digested with BamH1 and XbaI, and ligated together with a XbaI-Ndel fragment containing the SV40 3' UTR from vector pUAST-attB [19] into BamH1-Ndel-digested vector pUASPK10 [20]. GFP reporter plasmids containing md, m2 and m6 were generated by replacing the MluI-XbaI fragment containing the *bantam* hairpin by a MluI-XbaI-digested PCR fragment generated using oligos d1379 and d1380 from the corresponding existing donor plasmids.

PhiC31 integrase-mediated transgenesis was performed by the BestGene facility. Transgenes were targetted into the attP2 docking site of stock BL#8622.

Tissue-specific expression of the GFP-reporter transgenes in the female germline was induced using the *nos-Gal4-VP16* driver [21]. Ovaries were hand-dissected from 3 day-old females maintained on medium supplemented with dry yeast, fixed in 4% PFA and stained with 1 µg.mL⁻¹ DAPI following standard procedures, then mounted in Vectashield. Images were captured with a Zeiss Axioimager Apotome microscope.

## 2.7. Data and script availability

Small RNA-Seq data has been deposited at NCBI's SRA (https://www.ncbi.nlm.nih.gov/sra/) under BioProject accession numbers PRJNA668265 (for Fig. 1) and PRJNA841589 (for Fig. 4). Scripts and intermediary files are accessible at https://github.com/HKeyHKey/Busseau_et_al_2023 and https://www.igh.cnrs.fr/en/research/departments/genetics-development/systemic-impact-of-small-regulatory-rnas#programmes-informatiques.

# 3. Results

In an effort to generate a series of mutated alleles of the *Drosophila bantam* miRNA gene, we used CRISPR/Cas9-mediated genome editing to prepare three *Drosophila* lines where the *bantam* seed is mutated, and a fourth line where the *bantam* pre-miRNA hairpin is truncated (see Fig. 1A). In each seed mutant, miRNA* nucleotides facing the miRNA seed nucleotides were mutated too in order to preserve pre-miRNA secondary structure.

miRNA expression was then assessed by Small RNA-Seq in L1

larvae, where *bantam* is expressed at high levels [18]. We analyzed larvae heterozygous for the wild-type allele to provide an internal control. Biases in the propensity of various small RNAs to be deep-sequenced may distort their abundances in the Small RNA-Seq libraries [22,23]. In order to measure and correct such biases between wild-type and mutant *bantam*, RNA samples were mixed with spiked-in synthetic RNA oligos exhibiting the same sequence as the wild-type and intended mutant miRNA except for a central trinucleotide tag distinguishing them from miRNAs expressed from the larvae (see Fig. 1B).

The replacement of the *bantam* seed sequence (as well as facing *bantam* sequences, and the PAM located 9 nt downstream of the stem-loop) greatly reduced miRNA accumulation (compare wt to m2 *bantam* abundances for m2(proximal)/+ heterozygotes on Fig. 1C: 70% reduction for the mutant allele). It also perturbs the balance between miRNA isoforms: while wild-type *bantam* expresses mostly isoforms sharing the same 5' extremity, hence the same seed sequence, m2 *bantam* is expressed as a balanced mixture of isoforms with the intended CAAAAU seed and isoforms with alternative seed sequences (compare dark and pale purple on Fig. 1C). Because it is conceivable that the PAM mutation disrupts some unknown important sequence or structure motif, we prepared another version of the m2 edited hairpin, keeping that PAM unchanged and mutating another, distant PAM. That modification did not improve mutant miRNA accumulation (see m2 distal)/+ on Fig. 1C: 67% reduction for the mutant allele). In an attempt to reinforce miRNA/miRNA* asymmetry, we introduced an additional mutation, strengthening duplex base-pairing at the miRNA* 5' end [24-26]. While that mutation was predicted to increase miRNA accumulation, here it proved ineffective since m4 heterozygotes do not accumulate more mutant miRNA than m2 (see m4/+ on Fig. 1C: 85% reduction for the mutant allele).

We reasoned that the deleterious effect of the seed mutation may derive from a destabilization of the pre-miRNA stem double-helix: while the wild-type *bantam* seed contains 3 (C + G) and 3 (A + U) nucleotides, the mutant seed contains 1 (C + G) and 5 (A + U) nucleotides. In mammals, Drosha-mediated cleavage requires the double helix to be long enough, at least because its partner DGCR8 needs to bind to a stem of sufficient length [10]. It is therefore possible that a (A + U)-rich *bantam* seed decreases the pairing stability of the stem to the point that it is less efficiently recognized by the Drosha machinery.

We therefore aimed at designing a test to monitor the ability of candidate hairpins to be cleaved by Drosha in cultured cells, similar to the assay described by Ref. [27]. The insertion of the wild-type *bantam* hairpin in the 3' UTR of a firefly luciferase reporter inhibits reporter expression in S2R+ cells, relatively to the insertion of the truncated “md” hairpin, which is unsuitable for Drosha processing (see Fig. 2A and B). These results are in agreement with the notion that Drosha-mediated cleavage promotes degradation of the host transcript (reviewed in Ref. [28]), and provides the basis for a simple *ex vivo* assay to estimate the ability of candidate pri-miRNAs to serve as Drosha substrates.

We therefore compared 9 distinct mutant pre-miRNA sequences for their ability to repress a host luciferase reporter gene. Sequence and predicted secondary structure for these 9 variants are shown in Supplementary Fig. 1. We detected a significant reduction of luciferase expression for each of these candidates compared to the md hairpin, indicating that they all undergo some level of cleavage by Drosha (see Fig. 2B). When compared to the wild-type hairpin, every candidate was significantly less efficient at repressing their host reporter gene, except for the m6 construct (see Fig. 2B), suggesting that the m6 allele may be matured with an efficiency similar to that of wild-type *bantam*.

Consistent with the notion that inserted pre-miRNA hairpins
<figure><img src="image_4.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_5.png" /></figure>

<table><caption>C</caption><thead><tr><th></th><th>Empty</th><th>md</th><th>wt</th><th>m1</th><th>m2</th><th>m3</th><th>m4</th><th>m5</th><th>m6</th><th>m7</th><th>m8</th><th>m9</th></tr></thead><tbody><tr><td>Dunnett's test p-value:</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>reference = md:</td><td>&lt;2.2×10<sup>-16</sup></td><td>NA</td><td>&lt;2.2×10<sup>-16</sup></td><td>&lt;2.2×10<sup>-16</sup></td><td>&lt;2.2×10<sup>-16</sup></td><td>&lt;2.2×10<sup>-16</sup></td><td>&lt;2.2×10<sup>-16</sup></td><td>&lt;2.2×10<sup>-16</sup></td><td>&lt;2.2×10<sup>-16</sup></td><td>&lt;2.2×10<sup>-16</sup></td><td>&lt;2.2×10<sup>-16</sup></td><td>&lt;2.2×10<sup>-16</sup></td></tr><tr><td>reference = wt:</td><td>&lt;2.2×10<sup>-16</sup></td><td>&lt;2.2×10<sup>-16</sup></td><td>NA</td><td>1.6×10<sup>-14</sup></td><td>6.2×10<sup>-12</sup></td><td>3.6×10<sup>-4</sup></td><td>5.6×10<sup>-16</sup></td><td>4.4×10<sup>-3</sup></td><td>1</td><td>2.1×10<sup>-8</sup></td><td>1.1×10<sup>-8</sup></td><td>5.5×10<sup>-5</sup></td></tr></tbody></table>

<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Hairpin insertion-mediated inhibition of reporter expression is Drosha-dependent. **A.** Experimental procedure. Target genes were repressed by dsRNA-mediated RNAi, with repeated addition of dsRNA in the cell medium. The reporter plasmid (expressing firefly luciferase with various hairpins inserted in its 3' UTR, as well as a normalizing Renilla luciferase) was transfected 2 days before cell collection and reporter quantification. **B.** Firefly/Renilla luciferase activity ratio (y axis) depends on the identity of the hairpin inserted in firefly luciferase 3' UTR (md, wt, m2 or m6; on the x axis), and this effect is mostly lost when Drosha had been repressed by RNAi. An ANOVA analysis shows that hairpin identity (p = 3.6 × 10⁻⁹), dsRNA identity (p = 1.6 × 10⁻⁵) as well as the interaction between these two factors (p = 0.0030) have a significant effect. Two biological replicates of the reporter assay were prepared from a common culture of dsRNA-treated cells.</figcaption></figure>

inhibit reporter expression because of Drosha-mediated degradation of the reporter mRNA, we observed that this effect is essentially abolished when cells have been depleted for Drosha by RNAi (see Fig. 3). In a control experiment, we also depleted Exportin-5 by RNAi before transfecting the reporter plasmid. Exportin-5 (also known as RanBP21 in *Drosophila*) is required for nucleocytoplasmic export of pre-miRNAs in vertebrates [29–31], hence downstream of Drosha-mediated cleavage of the pri-miRNA. Surprisingly still, its knockdown also interfered with the inhibition of reporter expression by inserted hairpins (see Supplementary Fig. 2). This unexpected effect may be due to the role of *Drosophila* Exportin-5 in tRNA export, which could impact firefly and Renilla luciferase translation [32].

We also inserted *bantam* hairpin variants in a GFP host gene to assess their repressive effect *in vivo* in *Drosophila* ovaries (see Fig. 2C). The reporter with the uncleavable md variant produced high GFP both in the germarium and developing egg chambers, while the reporter with the m2 variant produced lower level of GFP in egg chambers (see Fig. 2C). In contrast, GFP from the reporters with wt *bantam* or the m6 variant was barely detectable both in the germarium and in developing egg chambers. These *in vivo* results mirror the *ex vivo* data, confirming that the presence of a m6 or wt hairpin in the 3' UTR of a reporter mRNA decreases its expression to a similar extent, while the m2 variant exerts a weaker repressive effect.

These results prompted us to prepare a *Drosophila* strain where the *bantam* gene has been mutated into the m6 variant (see Fig. 4A). Whole-body RNA from heterozygous L1 larvae was analyzed by Small RNA-Seq, using spiked-in RNA oligos for bias correction in each library. In order to take into account any potential difference in miRNA content across independent batches of ≈100 larvae, in this experiment we analyzed several replicates of larva batch. This design allowed us to not only compare the relative abundance of mutant and wild-type miRNA alleles within heterozygotes, but also to estimate the reproducibility of the observed fold-changes across independent batches of larvae. The availability of 10 spiked-in libraries also allowed us to verify the reproducibility of sequencing biases between the wild-type, m2 and m6 alleles of the *bantam* miRNA: ratios between the raw read counts for wt, m2 and m6 oligos proved consistent from library to library (see Supplementary Fig. 3).

In terms of abundance of miRNA isoforms with the correct seed sequence, expression of the m6 allele is very similar to that of the wild-type allele, and we were unable to detect a significant difference between the abundances of these two miRNA sequences (see Fig. 4B). The m6 allele therefore constitutes a successfully-edited allele, where the *bantam* seed has been mutated into a different hexamer without noticeably perturbing its accumulation level. The m2 allele, which was re-analyzed in parallel, shows a greater difference with the wild-type allele, although the observed
cleavage, loading on the effector protein, post-loading stability, ...), which can also be influenced by miRNA sequence, and which are not captured by our assay. In particular, accumulated miRNA/miRNA* ratios appear to differ between wild-type, m2 and m6 alleles (see Supplementary Fig. 4: miRNA/miRNA* ratio is ≈ 15 for the wild-type allele, and ≈3 and ≈6 for m2 and m6 alleles respectively). This observation shows that the efficiency of Drosha-mediated maturation is one factor among others, controlling the accumulation of seed-edited miRNAs.

We also observed that the mutation of the miRNA seed sequence (together with that of facing miRNA* nucleotides) affects the distribution of miRNA isoforms. While wild-type *bantam* harbors a very homogeneous 5' end (yielding mature molecules which have mostly the same seed sequence), we note that m2 and m6 mutants tend to generate more heterogeneous 5' ends (see Figs. 1C and 4B). This result reveals a subtle dependence of Dicer cleavage site selection, of miRNA loading efficiency, or of miRNA stability, to miRNA sequence, even without any change in the predicted hairpin secondary structure. In the case of the *bantam* m6 mutant, a simultaneous increase in pan-isoform miRNA accumulation compensates for this effect, therefore achieving a highly accurate wild-type level of accumulation for the m6 isoforms with correct seed sequence. Further analyses will be required to characterize more finely such subtle consequences of sequence editing on various aspects of miRNA biogenesis and accumulation.

## 5. Conclusions

Editing *bantam*'s seed sequence triggered unwanted perturbation of miRNA biogenesis *in vivo*, even though facing miRNA* nucleotides were simultaneously mutated in an attempt to preserve pre-miRNA secondary structure. Screening candidate edited pre-miRNA sequences in a cultured cell assay helped identifying the best processed candidate, therefore allowing us to generate an edited *Drosophila* line where the *bantam* seed has been mutated without affecting its accumulation level. Usage of this assay could be generalized, to screen candidate mutant pre-miRNAs for their ability to be efficiently processed.

## Author contribution

I. B. and H. Seitz conceived the project; I. B., É. H. and H. Somaï collected data; S. M. and H. Seitz analyzed data; I. B. and H. Seitz wrote the manuscript. All authors have approved the final article.

## Acknowledgments

We are grateful to Simon Nicot for guidance in cell culture, Lara Demont for help in the screening of edited flies, Laly Robledo for her implication in the setting of Luciferase assays, and Guillaume Canal for his help in optimizing spike-in oligo sequences. We acknowledge the MRI imaging facility, member of the France-Biolmaging infrastructure, and the MGX sequencing facility, member of the France Génomique National Infrastructure, supported respectively by contracts ANR-10-INBS-04 and ANR-10-INBS-09 "Investments for the future" of French National Research Agency, as well as the Biocampus *Drosophila* facility. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. This work was funded by the CNRS.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biochi.2023.06.007.

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