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RNA Biology Volume 21, 2024 - Issue 1
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Research Paper

MiRNAs differentially expressed in vegetative and reproductive organs of Marchantia polymorpha – insights into their expression pattern, gene structures and function

Bharti Aggarwala Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, PolandORCID Iconhttps://orcid.org/0000-0003-1127-6566View further author information
ORCID Icon,
Wojciech Maciej Karlowskib Department of Computational Biology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, PolandORCID Iconhttps://orcid.org/0000-0002-8086-5404View further author information
ORCID Icon,
Przemyslaw Nuca Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, PolandORCID Iconhttps://orcid.org/0000-0002-8380-1166View further author information
ORCID Icon,
Artur Jarmolowskia Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, PolandORCID Iconhttps://orcid.org/0000-0002-1073-146XView further author information
ORCID Icon,
Zofia Szweykowska-Kulinskaa Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, PolandCorrespondence[email protected]
ORCID Iconhttps://orcid.org/0000-0003-0032-469XView further author information
ORCID Icon &
Halina Pietrykowskaa Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, PolandCorrespondence[email protected]
ORCID Iconhttps://orcid.org/0000-0003-0524-8067View further author information
ORCID Icon
Pages 1-12 | Accepted 05 Jan 2024, Published online: 01 Feb 2024

Figures & data

Figure 1. Accumulation of conserved miRNAs in M. polymorpha vegetative and reproductive organs. (A-G) sRNA NGS data (left panels) and northern blot hybridization results (right panels) from male vegetative thalli (Mv), antheridiophores (Ma), female vegetative thalli (Fv) and archegoniophores (Fa); normalized read counts are presented above each bar; RPM-reads per million; U6snRNA was used as RNA loading control; the numbers below blot images are the relative intensities of miRNA bands; control signals in each blot were treated as 1; differences in signal intensity were calculated separately for male vegetative thalli control/antheridiophores, female vegetative thalli control/archegoniophores; the left side of northern blots shows the RNA marker depicting 17–25 nucleotide long RNAs.

Figure 1. Accumulation of conserved miRNAs in M. polymorpha vegetative and reproductive organs. (A-G) sRNA NGS data (left panels) and northern blot hybridization results (right panels) from male vegetative thalli (Mv), antheridiophores (Ma), female vegetative thalli (Fv) and archegoniophores (Fa); normalized read counts are presented above each bar; RPM-reads per million; U6snRNA was used as RNA loading control; the numbers below blot images are the relative intensities of miRNA bands; control signals in each blot were treated as 1; differences in signal intensity were calculated separately for male vegetative thalli control/antheridiophores, female vegetative thalli control/archegoniophores; the left side of northern blots shows the RNA marker depicting 17–25 nucleotide long RNAs.

Figure 2. Accumulation profile of liverwort-specific MpmiR11737a, its pri-miRNA level, gene structure and target analyses. (A) sRNA NGS sequencing results; normalized read counts are presented above each bar; RPM-reads per million; (B) Northern blot hybridization analysis; U6snRNA was used as RNA loading control; the numbers below blot images are the relative intensities of miRNA bands; control signals in each blot were treated as 1; differences in signal intensity were calculated separately for male vegetative thalli control/antheridiophores, female vegetative thalli control/archegoniophores (numbers in the first row) or antheridiophores control/archegoniophores (numbers in the second row); the left side of northern blots shows the RNA marker depicting 17–25 nucleotide long RNAs; (C) RT-qPCR expression level of pri-MpmiR11737a; *p-value < 0.05; (D) Hairpin structure of pre-MpmiR11737a; nucleotides highlighted in gray represent MpmiR11737a sequence; the minimum free energy (∆G) of predicted structure is shown on the right side (E) MpMIR11737a gene structure (upper panel; pri-miRNA – light gray; pre-miRNA-dark gray), Mp5g12920 gene overlapping with MpMIR11737a gene (lower panel); boxes – exons (UTRs – striped; CDS – white); lines – introns; scale bar corresponds to 1kb; (F) Target plot (T-plot) of target mRNA Mp1g15010 based on degradome data; red arrow points to the mRNA cleavage site; T-plot is accompanied by a duplex of miRNA and its target mRNA; nucleotide marked in bold points to the mRNA cleavage site; (G) RT- qPCR expression level of Mp1g15010; ***p-value < 0.001; male vegetative thalli (Mv), antheridiophores (Ma), female vegetative thalli (Fv) and archegoniophores (Fa).

Figure 2. Accumulation profile of liverwort-specific MpmiR11737a, its pri-miRNA level, gene structure and target analyses. (A) sRNA NGS sequencing results; normalized read counts are presented above each bar; RPM-reads per million; (B) Northern blot hybridization analysis; U6snRNA was used as RNA loading control; the numbers below blot images are the relative intensities of miRNA bands; control signals in each blot were treated as 1; differences in signal intensity were calculated separately for male vegetative thalli control/antheridiophores, female vegetative thalli control/archegoniophores (numbers in the first row) or antheridiophores control/archegoniophores (numbers in the second row); the left side of northern blots shows the RNA marker depicting 17–25 nucleotide long RNAs; (C) RT-qPCR expression level of pri-MpmiR11737a; *p-value < 0.05; (D) Hairpin structure of pre-MpmiR11737a; nucleotides highlighted in gray represent MpmiR11737a sequence; the minimum free energy (∆G) of predicted structure is shown on the right side (E) MpMIR11737a gene structure (upper panel; pri-miRNA – light gray; pre-miRNA-dark gray), Mp5g12920 gene overlapping with MpMIR11737a gene (lower panel); boxes – exons (UTRs – striped; CDS – white); lines – introns; scale bar corresponds to 1kb; (F) Target plot (T-plot) of target mRNA Mp1g15010 based on degradome data; red arrow points to the mRNA cleavage site; T-plot is accompanied by a duplex of miRNA and its target mRNA; nucleotide marked in bold points to the mRNA cleavage site; (G) RT- qPCR expression level of Mp1g15010; ***p-value < 0.001; male vegetative thalli (Mv), antheridiophores (Ma), female vegetative thalli (Fv) and archegoniophores (Fa).

Figure 3. Accumulation profile of liverwort-specific MpmiR11737b, its pri-miRNA level and host gene structure. (A) sRNA NGS sequencing results; normalized read counts are presented above each bar; RPM-reads per million; (B) RT-qPCR expression level of pri- MpmiR11737b; *p-value < 0.05; (C) Hairpin structure of pre-MpmiR11737b; nucleotides highlighted in gray represent MpmiR11737b sequence; the minimum free energy (∆G) of predicted structure is shown on the right side (D) pre-MpmiR11737b (upper panel; dark gray), Mp8g07030 gene hosting pre-MpmiR11737b (lower panel); boxes – exons (UTRs – striped; CDS – white); lines – introns; scale bar corresponds to 1kb; male vegetative thalli (Mv), antheridiophores (Ma), female vegetative thalli (Fv) and archegoniophores (Fa).

Figure 3. Accumulation profile of liverwort-specific MpmiR11737b, its pri-miRNA level and host gene structure. (A) sRNA NGS sequencing results; normalized read counts are presented above each bar; RPM-reads per million; (B) RT-qPCR expression level of pri- MpmiR11737b; *p-value < 0.05; (C) Hairpin structure of pre-MpmiR11737b; nucleotides highlighted in gray represent MpmiR11737b sequence; the minimum free energy (∆G) of predicted structure is shown on the right side (D) pre-MpmiR11737b (upper panel; dark gray), Mp8g07030 gene hosting pre-MpmiR11737b (lower panel); boxes – exons (UTRs – striped; CDS – white); lines – introns; scale bar corresponds to 1kb; male vegetative thalli (Mv), antheridiophores (Ma), female vegetative thalli (Fv) and archegoniophores (Fa).

Figure 4. Accumulation profile of liverwort-specific MpmiR11865*/MpmiR11865, its pri-miRNA level, gene structure and targets analyses. (A) Hairpin structure of pre-MpmiR11865*/MpmiR11865; nucleotides highlighted in dark gray represent MpmiR11865* sequence and in light gray MpmiR11865 sequence; the minimum free energy (∆G) of predicted structure is shown on the right side of the structure; (B) RT-qPCR expression level of pri-MpmiR11865*/MpmiR11865; *p-value < 0.05; (C) MpMIR11865 gene structure (pri-miRNA – light gray; pre-miRNA-dark gray); scale bar corresponds to 1kb; (D) MpmiR11865* sRNA NGS sequencing results (left side); normalized read counts are presented above each bar; RPM-reads per million; northern blot hybridization analysis (right side): U6snRNA was used as RNA loading control; the numbers below blot images are the relative intensities of miRNA bands; control signals in each blot were treated as 1; differences in signal intensity were calculated separately for male vegetative thalli control/antheridiophores, female vegetative thalli control/archegoniophores (numbers in the first row) or antheridiophores control/archegoniophores (numbers in the second row); the left side of northern blots shows the RNA marker depicting 17–25 nucleotide long RNAs; (E) Target plot (T-plot) of MpmiR11865* target mRNA (Mp1g05970) based on degradome data (left side); red arrow points to the mRNA cleavage site; T-plot is accompanied by a duplex of miRNA and its target mRNA; nucleotide marked in bold points to the mRNA cleavage site; RT-qPCR expression level of Mp1g05970 (right side); *p- value < 0.05; **p-value < 0.01; (F) Graph represents MpmiR11865 sRNA NGS sequencing results (left side); normalized read counts are presented above each bar; RPM-reads per million; northern blot hybridization analysis (right side): U6snRNA was used as RNA loading control; the numbers below blot images are the relative intensities of miRNA bands; control signals in each blot were treated as 1; differences in signal intensity were calculated separately for male vegetative thalli control/antheridiophores, female vegetative thalli control/archegoniophores; the left side of northern blot shows the RNA marker depicting 19–23 nucleotide long RNAs; (G) Target plot (T-plot) of MpmiR11865 target mRNA (Mp6g13460) based on degradome data (left side); red arrow points to the mRNA cleavage site; T-plot is accompanied by a duplex of miRNA and its target mRNA; nucleotide marked in bold points to the mRNA cleavage site; RT-qPCR expression level of Mp6g13460 (right side); *p-value < 0.05; antheridiophores (Ma), male vegetative thalli (Mv), archegoniophores (Fa) and female vegetative thalli (Fv).

Figure 4. Accumulation profile of liverwort-specific MpmiR11865*/MpmiR11865, its pri-miRNA level, gene structure and targets analyses. (A) Hairpin structure of pre-MpmiR11865*/MpmiR11865; nucleotides highlighted in dark gray represent MpmiR11865* sequence and in light gray MpmiR11865 sequence; the minimum free energy (∆G) of predicted structure is shown on the right side of the structure; (B) RT-qPCR expression level of pri-MpmiR11865*/MpmiR11865; *p-value < 0.05; (C) MpMIR11865 gene structure (pri-miRNA – light gray; pre-miRNA-dark gray); scale bar corresponds to 1kb; (D) MpmiR11865* sRNA NGS sequencing results (left side); normalized read counts are presented above each bar; RPM-reads per million; northern blot hybridization analysis (right side): U6snRNA was used as RNA loading control; the numbers below blot images are the relative intensities of miRNA bands; control signals in each blot were treated as 1; differences in signal intensity were calculated separately for male vegetative thalli control/antheridiophores, female vegetative thalli control/archegoniophores (numbers in the first row) or antheridiophores control/archegoniophores (numbers in the second row); the left side of northern blots shows the RNA marker depicting 17–25 nucleotide long RNAs; (E) Target plot (T-plot) of MpmiR11865* target mRNA (Mp1g05970) based on degradome data (left side); red arrow points to the mRNA cleavage site; T-plot is accompanied by a duplex of miRNA and its target mRNA; nucleotide marked in bold points to the mRNA cleavage site; RT-qPCR expression level of Mp1g05970 (right side); *p- value < 0.05; **p-value < 0.01; (F) Graph represents MpmiR11865 sRNA NGS sequencing results (left side); normalized read counts are presented above each bar; RPM-reads per million; northern blot hybridization analysis (right side): U6snRNA was used as RNA loading control; the numbers below blot images are the relative intensities of miRNA bands; control signals in each blot were treated as 1; differences in signal intensity were calculated separately for male vegetative thalli control/antheridiophores, female vegetative thalli control/archegoniophores; the left side of northern blot shows the RNA marker depicting 19–23 nucleotide long RNAs; (G) Target plot (T-plot) of MpmiR11865 target mRNA (Mp6g13460) based on degradome data (left side); red arrow points to the mRNA cleavage site; T-plot is accompanied by a duplex of miRNA and its target mRNA; nucleotide marked in bold points to the mRNA cleavage site; RT-qPCR expression level of Mp6g13460 (right side); *p-value < 0.05; antheridiophores (Ma), male vegetative thalli (Mv), archegoniophores (Fa) and female vegetative thalli (Fv).

Figure 5. Accumulation profile of liverwort-specific MpmiR11887, its pri-miRNA level, gene structure and target analyses. (A) sRNA NGS sequencing results; normalized read counts are presented above each bar; RPM-reads per million; (B) Northern blot hybridization analysis; U6snRNA was used as RNA loading control; the left side of northern blots shows the RNA marker depicting 17–25 nucleotide long RNAs; (C) Hairpin structure of pre-MpmiR11887; nucleotides highlighted in gray represent MpmiR11887 sequence; the minimum free energy (∆G) of predicted structure is shown above the precursor; (D) RT-qPCR expression level of pri-MpmiR11887; *p-value < 0.05; (E) MpMIR11887 gene structure (upper panel; pri-miRNA – light gray; pre-miRNA-dark gray), putative Mp6g01830 protein-coding gene overlapping with MpMIR11887 gene (lower panel); boxes – exons (UTRs – striped; CDS – white); scale bar corresponds to 1kb; (F) Target plot (T-plot) of target mRNA Mp1g20730 based on degradome data; red arrow points to the mRNA cleavage site; T-plot is accompanied by a duplex of miRNA and its target mRNA; nucleotide marked in bold points to the mRNA cleavage site; (G) RT-qPCR expression level of Mp1g20730; *p-value < 0.05; male vegetative thalli (Mv), antheridiophores (Ma), female vegetative thalli (Fv) and archegoniophores (Fa).

Figure 5. Accumulation profile of liverwort-specific MpmiR11887, its pri-miRNA level, gene structure and target analyses. (A) sRNA NGS sequencing results; normalized read counts are presented above each bar; RPM-reads per million; (B) Northern blot hybridization analysis; U6snRNA was used as RNA loading control; the left side of northern blots shows the RNA marker depicting 17–25 nucleotide long RNAs; (C) Hairpin structure of pre-MpmiR11887; nucleotides highlighted in gray represent MpmiR11887 sequence; the minimum free energy (∆G) of predicted structure is shown above the precursor; (D) RT-qPCR expression level of pri-MpmiR11887; *p-value < 0.05; (E) MpMIR11887 gene structure (upper panel; pri-miRNA – light gray; pre-miRNA-dark gray), putative Mp6g01830 protein-coding gene overlapping with MpMIR11887 gene (lower panel); boxes – exons (UTRs – striped; CDS – white); scale bar corresponds to 1kb; (F) Target plot (T-plot) of target mRNA Mp1g20730 based on degradome data; red arrow points to the mRNA cleavage site; T-plot is accompanied by a duplex of miRNA and its target mRNA; nucleotide marked in bold points to the mRNA cleavage site; (G) RT-qPCR expression level of Mp1g20730; *p-value < 0.05; male vegetative thalli (Mv), antheridiophores (Ma), female vegetative thalli (Fv) and archegoniophores (Fa).

Figure 6. Accumulation profile of liverwort-specific MpmiR11796, its pri-miRNA level, gene structure and target analyses. (A) sRNA NGS sequencing results; normalized read counts are presented above each bar; RPM-reads per million; (B) Northern blot hybridization analysis; U6snRNA was used as RNA loading control; the numbers below blot images are the relative intensities of miRNA bands; control signals in each blot were treated as 1; differences in signal intensity were calculated separately for male vegetative thalli control/antheridiophores, female vegetative thalli control/archegoniophores (numbers in the first row) or antheridiophores control/archegoniophores (numbers in the second row); the left side of northern blots shows the RNA marker depicting 17–25 nucleotide long RNAs; (C) Hairpin structure of pre-MpmiR11796; nucleotides highlighted in gray represent MpmiR11796 sequence; the minimum free energy (∆G) of predicted structure is shown below the precursor; (D) RT-qPCR expression level of pri-MpmiR11796; *p-value < 0.05; (E) MpMIR11796 gene structure (upper panel; pri-miRNA – light gray; pre-miRNA-dark gray), Mp4g11670 gene overlapping with MpMIR11796 gene (lower panel); boxes – exons (UTRs – striped; CDS – white); lines – introns; scale bar corresponds to 1kb; (F) Target plot (T-plot) of target mRNA Mp4g20750 based on degradome data; red arrow points to the mRNA cleavage site; T-plot is accompanied by a duplex of miRNA and its target mRNA; nucleotide marked in bold points to the mRNA cleavage site; (G) RT- qPCR expression level of Mp4g20750; **p-value < 0.01; male vegetative thalli (Mv), antheridiophores (Ma), female vegetative thalli (Fv) and archegoniophores (Fa).

Figure 6. Accumulation profile of liverwort-specific MpmiR11796, its pri-miRNA level, gene structure and target analyses. (A) sRNA NGS sequencing results; normalized read counts are presented above each bar; RPM-reads per million; (B) Northern blot hybridization analysis; U6snRNA was used as RNA loading control; the numbers below blot images are the relative intensities of miRNA bands; control signals in each blot were treated as 1; differences in signal intensity were calculated separately for male vegetative thalli control/antheridiophores, female vegetative thalli control/archegoniophores (numbers in the first row) or antheridiophores control/archegoniophores (numbers in the second row); the left side of northern blots shows the RNA marker depicting 17–25 nucleotide long RNAs; (C) Hairpin structure of pre-MpmiR11796; nucleotides highlighted in gray represent MpmiR11796 sequence; the minimum free energy (∆G) of predicted structure is shown below the precursor; (D) RT-qPCR expression level of pri-MpmiR11796; *p-value < 0.05; (E) MpMIR11796 gene structure (upper panel; pri-miRNA – light gray; pre-miRNA-dark gray), Mp4g11670 gene overlapping with MpMIR11796 gene (lower panel); boxes – exons (UTRs – striped; CDS – white); lines – introns; scale bar corresponds to 1kb; (F) Target plot (T-plot) of target mRNA Mp4g20750 based on degradome data; red arrow points to the mRNA cleavage site; T-plot is accompanied by a duplex of miRNA and its target mRNA; nucleotide marked in bold points to the mRNA cleavage site; (G) RT- qPCR expression level of Mp4g20750; **p-value < 0.01; male vegetative thalli (Mv), antheridiophores (Ma), female vegetative thalli (Fv) and archegoniophores (Fa).
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Data availability statement

The data that support the findings of this study are available in the National Center for Biotechnology Information (NCBI) Sequence Read Archive database at https://www.ncbi.nlm.nih.gov/bioproject/1008567

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