Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Apr 26;39(4):110745.
doi: 10.1016/j.celrep.2022.110745.

Critical contribution of 3' non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA

Ye Duan  1 , Isana Veksler-Lublinsky  2 , Victor Ambros  3
Affiliations

Critical contribution of 3' non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA

Ye Duan et al. Cell Rep. .

Abstract

Base pairing of the seed region (g2-g8) is essential for microRNA targeting; however, the in vivo function of the 3' non-seed region (g9-g22) is less well understood. Here, we report a systematic investigation of the in vivo roles of 3' non-seed nucleotides in microRNA let-7a, whose entire g9-g22 region is conserved among bilaterians. We find that the 3' non-seed sequence functionally distinguishes let-7a from its family paralogs. The complete pairing of g11-g16 is essential for let-7a to fully repress multiple key targets, including evolutionarily conserved lin-41, daf-12, and hbl-1. Nucleotides at g17-g22 are less critical but may compensate for mismatches in the g11-g16 region. Interestingly, a certain minimal complementarity to let-7a 3' non-seed sequence can be required even for sites with perfect seed pairing. These results provide evidence that the specific configurations of both seed and 3' non-seed base pairing can critically influence microRNA-mediated gene regulation in vivo.

Keywords: 3′ non-seed pairing; CP: Molecular biology; daf-12; let-7; lin-41; microRNA; post-transcriptional regulation.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. let-7a sequence is conserved across bilaterian species
(A) Summary of let-7 family miRNAs across bilaterian phylogeny. Bar length, number of let-7 family isoforms; bar color, sequence distance relative to hsa-let-7a-5p. (B) Nucleotide frequency of the let-7 family isoforms most similar to hsa-let-7a-5p across bilaterians. See also Figure S1.
Figure 2.
Figure 2.. The 3′ non-seed sequence determines the functional specificity of let-7a among paralogs
(A) Strategy for generation of let-7(ma341) by CRISPR-Cas9-mediated swap of the pre-let-7 sequence for the pre-mir-84 sequence. Dot-bracket notations show the pre-miRNA structures predicted by RNAfold (Denman, 1993). (B–E) Developmental profiles of miR-84 and let-7a miRNAs by Fireplex assay. Expression is calibrated with synthetic miR-84 and let-7a oligos. Time, hours after feeding starvation-arrested L1 larvae. (F) Representative lineages and COL-19::GFP expression patterns of V1–V4 seam cells. (G) Seam cell numbers of young adults. n, numbers of animals tested. (H and I) Representative expression patterns of COL-19::GFP in adults. Scale bars, 100 μm. (J and K) Differential interference contrast (DIC) images of the vulva region of adults. Scale bars, 25 μm. (L) Adult lethality for wild type (WT), let-7(ma341), let-7(n2853, G5C), and let-7(ma393, null). Lethal phenotypes are categorized as severe (via bursting of young adults through the vulva) or mild (via matricide of egg-laying-defective adults). The data in (B)–(E) represent 3 biological replicas. Error bars indicate mean ± SD. See also Figure S5. Details of the phenotypes are available in Table S1.
Figure 3.
Figure 3.. Contribution of single 3′ non-seed nucleotides to the in vivo function of let-7a
(A) Alignment of pre-miRNA sequences. Boxed 3′ non-seed regions. (B) Small RNA sequencing reads from the L4 larvae that mapped to WT or mutant let-7a sequences for each single mutant. The reads mapping to WT let-7a for strains carrying mutations at g11–g13 include WT let-7a miRNA from balancer umnIs25(mnDp1). The WT reads from U21A/U22A mutants likely reflect let-7a(mutant) miRNAs whose 3′ ends had been trimmed and subsequently uridylated in vivo, resulting in artificial let-7a(+) reads. Dashed lines, 100% (top) and 50% (bottom) of RPM of let-7a(+) in WT. (C) Quantitation of let-7a lf phenotypes: percentage of animals with abnormal COL-19:GFP expression (top), numbers of progeny per animal (center), and percentage of adult lethality (bottom). Lethal phenotypes are categorized as in Figure 2L. Abnormal COL-19:GFP patterns are classified as no Hyp7 expression (severe) or faint Hyp7 expression (mild). (D–G) DIC images of the vulva region in adults at 25°C. Scale bars, 25 μm. (H) Functional synergy between g18 and the critical non-seed region based on vulva integrity defect. Labels are identical to (C) (bottom). (I) Summary of the functional merits of let-7a 3′ non-seed nucleotides. Colored circle, 3′ non-seed nucleotide; red proportion, average adult lethality caused by vulva bursting at all temperatures. Error bars indicate mean ± SD. See also Figures S2 and S3. Details of the phenotypes are available in Table S1.
Figure 4.
Figure 4.. The let-7a critical non-seed nucleotides confer in vivo function by repressing lin-41 and additional 3′ targets
(A) Pairing configurations between let-7a and lin-41 LCS1/2. (B–E) DIC images of adult vulva regions representative of the WT lin-41(tn1541) (B), lin-41(tn1541);let-7(ma432) (C), lin-41(tn1541ma480);let-7(ma432) (D) and lin-41(tn1541ma480) (E) at 25°C. Scale bars, 25 μm. All of the genotypes tested in this figure and in Figure 5 and Figure 7 include a GFP tag on LIN-41, denoted as tn1541 (Spike et al., 2014). (F) Vulva integrity defects reflected by adult lethality (bottom) and the number of progeny (top). Lethal phenotypes are categorized identically to Figure 2L. (G) Representative COL-19::GFP pattern in young adults at 25°C. Scale bars, 100 μm. (H) Models proposing that lin-41 and additional 3′ targets are de-repressed in let-7a non-seed mutants. See also Figure S4. Details of the phenotypes are available in Table S1.
Figure 5.
Figure 5.. let-7a represses multiple targets, including daf-12 and hbl-1, through 3′ non-seed pairing
(A) Differential expression analysis of translatomes of lin-41(tn1541ma480);let-7(ma432) and lin-41(tn1541). Hollow points, developmentally dynamic genes for which the observed perturbation could be caused by asynchrony in staging between samples, independently of genotype; solid points, genes for which the observed perturbation is judged to likely reflect an effect of the let-7a mutation specifically (see STAR Methods; Table S3). Circular points with text labels, genes with predicted let-7a 3′ sites; triangle points, genes containing only let-7a seed-only sites. (B) Retarded COL-19::GFP patterns characteristic of lin-41(tn1541ma480);let-7(ma432) young adults at 25°C under empty vector, daf-12(RNAi), or hbl-1(RNAi). Scale bars, 100 μm. (C) Fold changes of mRNA abundance and translational efficiency (TE). Red points, genes with significantly increased mRNA abundance. Orange points, genes with significantly increased TE. Purple points, genes with both significantly increased TE and mRNA abundance. Circled points, genes with significantly increased RPFs in (A). Predicted let-7a 3′ targets are text labeled. (D and E) DIC (left) and fluorescent (right) microscopy images showing expression of DAF-12::mSCARLET at L3/L4 stages at 25°C representative of lin-41(tn1541);daf-12(ma498) (D) and lin-41(tn1541);daf-12(ma498ma568) (E). Scale bars, 5 μm. All of the fluorescent images were generated with identical exposure and processing conditions. (F) Quantification of relative fluorescent intensity of DAF-12::mSCARLET in seam cell nuclei relative to the adjacent Hyp7 nuclei. The numbers of seam cells quantified are shown under each violin plot. A total of 22 and 28 animals were scored for daf-12(ma498) at L3 and L4, respectively; 23 and 31 animals were scored for daf-12(ma498ma568) at L3 and L4, respectively. (G) Models for daf-12 repression at L3/L4. Purple region, trinucleotide mutations that disrupt duplexing with g11–g13 of let-7a. (H) Representative expression patterns of COL-19:GFP in adult animals. Scale bars, 100 μm. Data in (A) and (C) represent 3 biological replicas. See also Figures S6 and S7.
Figure 6.
Figure 6.. Critical 3′ non-seed pairing and seed pairing together contribute to let-7a target repression
(A–F) Pairing configurations of let-7a to the lin-41 LCSs and associated phenotypes for lin-41(ma501) (A), lin-41(ma501ma480) (B), lin-41(ma501ma545) (C), lin-41(+) (D), lin-41(ma480) (E), and lin-41(ex11) (F), arranged in a matrix based on mismatches in seed (vertical axis) and critical non-seed region (horizontal axis). Each panel includes pairing pattern (top left), vulva defect (young adult lethality and number of progeny, top right), and heterochronic phenotype (bottom). Scale bars, 100 μm. (G and H) Vulva integrity of lin-41 LCSs mutants with 3′ pairing mismatches in the context of imperfect (G) and perfect seed pairing (H). (I) Models depicting the minimal pairing requirement for 3′ compensatory and 3′ supplemental effects. Statistical significance indicates relative to WT. Phenotypes were tested at 25°C. All of the lin-41 alleles tested in this figure do not contain a fluorescent protein tag. Details of the phenotypes available in Table S1.
Figure 7.
Figure 7.. Perfect seed pairing alone is insufficient for let-7a family paralogs to confer full repression of lin-41
(A) Predicted seed-only pairing (RNAhybrid (Rehmsmeier et al., 2004) of let-7a and paralogs miR-48/84/241 to LCS1 (top) and LCS2 (bottom) of lin-41(ma501). Requirements for minimal supplemental pairing (t11–t12 pairing or t13–t16 pairing; Figure 6I) are indicated by brown or purple. (B) Vulva integrity defects based on adult lethality (top) and number of progeny (bottom) in lin-41(tn1541), lin-41(tn1541);let-7(ma393,null) and lin-41(tn1541ma501);let-7(ma393). (C) Predicted targeting configurations. Details of the phenotypes available in Table S1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abbott AL, Alvarez-Saavedra E, Miska EA, Lau NC, Bartel DP, Horvitz HR, and Ambros V (2005). The let-7 MicroRNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans. Dev. Cell 9, 403–414. - PMC - PubMed
    1. Abrahante JE, Daul AL, Li M, Volk ML, Tennessen JM, Miller EA, and Rougvie AE (2003). The Caenorhabditis elegans hunchback-like gene lin-57/hbl-1 controls developmental time and is regulated by microRNAs. Dev. Cell 4, 625–637. - PubMed
    1. Aeschimann F, Kumari P, Bartake H, Gaidatzis D, Xu L, Ciosk R, and Grosshans H (2017). LIN41 post-transcriptionally silences mRNAs by two distinct and position-dependent mechanisms. Mol. Cell 65, 476–489.e4. - PubMed
    1. Aeschimann F, Neagu A, Rausch M, and Grosshans H (2019). let-7 coordinates the transition to adulthood through a single primary and four secondary targets. Life Sci. Alliance 2, e201900335. - PMC - PubMed
    1. Aeschimann F, Xiong J, Arnold A, Dieterich C, and Grosshans H (2015). Transcriptome-wide measurement of ribosomal occupancy by ribosome profiling. Methods 85, 75–89. - PubMed

Publication types