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Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis.

Köster T, Meyer K, Weinholdt C, Smith LM, Lummer M, Speth C, Grosse I, Weigel D, Staiger D - Nucleic Acids Res. (2014)

Bottom Line: AtGRP7 overexpression caused a significant reduction in the level of 30 miRNAs and an increase for 14 miRNAs with a minimum log2 fold change of ± 0.5.Mutation of an arginine in the RNA recognition motif abrogated in vivo binding and the effect on miRNA and pri-miRNA levels, indicating that AtGRP7 inhibits processing of these pri-miRNAs by direct binding.Thus, AtGRP7 is an hnRNP-like protein with a role in processing of pri-miRNAs in addition to its role in pre-mRNA splicing.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Physiology, Bielefeld University.

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AtGRP7 affects alternative splicing of pri-miRNAs. (A) Scheme of MIR172b. Black boxes = exons, grey box = position of the pri-miRNA, thin line = introns. The arrows denote the position of the primers used in (B). (B) RNA from the AtGRP7-ox lines D and G and Col-2 wt was analysed by RT-PCR. The amplification products corresponding to the intron-retained form and the fully spliced forms are indicated by arrowheads and asterisks, respectively. PP2A served as a control. DNA = genomic DNA. (C) The ratio of intron-retained versus spliced pri-miR172b was quantified using Bioanalyzer DNA1000 chips. Shown is the mean of two reps. (D) Scheme of the non-protein-coding RNA harbouring MIR162a. Black boxes = exons, grey box = position of the pri-miRNA, open boxes = annotated 5′and 3′UTRs, thin line = introns. The arrows denote the position of the primers used in (E). (E) RNA from the AtGRP7-ox lines D and G and Col-2 wt was analysed by RT-PCR. The transcript forms corresponding to the amplification products are indicated. The rhombus denotes an alternative version of the 168 nt band generated by an alternative 3′splice site 3 nt downstream of the authentic 3′splice site. PP2A served as a control. DNA = genomic DNA. (F) The ratio of the alternative splice forms versus the spliced form was quantified using Bioanalyzer DNA1000 chips. Shown is the mean of two reps.
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Figure 6: AtGRP7 affects alternative splicing of pri-miRNAs. (A) Scheme of MIR172b. Black boxes = exons, grey box = position of the pri-miRNA, thin line = introns. The arrows denote the position of the primers used in (B). (B) RNA from the AtGRP7-ox lines D and G and Col-2 wt was analysed by RT-PCR. The amplification products corresponding to the intron-retained form and the fully spliced forms are indicated by arrowheads and asterisks, respectively. PP2A served as a control. DNA = genomic DNA. (C) The ratio of intron-retained versus spliced pri-miR172b was quantified using Bioanalyzer DNA1000 chips. Shown is the mean of two reps. (D) Scheme of the non-protein-coding RNA harbouring MIR162a. Black boxes = exons, grey box = position of the pri-miRNA, open boxes = annotated 5′and 3′UTRs, thin line = introns. The arrows denote the position of the primers used in (E). (E) RNA from the AtGRP7-ox lines D and G and Col-2 wt was analysed by RT-PCR. The transcript forms corresponding to the amplification products are indicated. The rhombus denotes an alternative version of the 168 nt band generated by an alternative 3′splice site 3 nt downstream of the authentic 3′splice site. PP2A served as a control. DNA = genomic DNA. (F) The ratio of the alternative splice forms versus the spliced form was quantified using Bioanalyzer DNA1000 chips. Shown is the mean of two reps.

Mentions: AtGRP7 overexpression leads to reduced levels of miR172b and miR172b* with a concomitant increase in pri-miR172b which contains introns both 5′ and 3′ of the stem–loop structure (Figure 6A). Because splicing of introns located 3′ of the stem–loop can impact accumulation of mature miRNAs (73,74), we asked whether AtGRP7 affects splicing of pri-miR172b. In wt plants, some pri-miR172b retaining the intron was detected in addition to the spliced form. In AtGRP7-ox plants, the spliced pri-miR172b accumulated at the expense of the intron retained form, indicating that AtGRP7 indeed affects splicing of pri-miRNAs (Figure 6B and C). In contrast, splicing of the introns located upstream of the hairpin was not altered (not shown). Thus, an enhanced assembly of the spliceosome at the 5′end of the downstream intron in AtGRP7-ox plants may interfere with pri-miRNA processing. Alternatively, a slower release of miR172b and miR172b* caused by AtGRP7 binding may allow more efficient assembly of the spliceosome.


Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis.

Köster T, Meyer K, Weinholdt C, Smith LM, Lummer M, Speth C, Grosse I, Weigel D, Staiger D - Nucleic Acids Res. (2014)

AtGRP7 affects alternative splicing of pri-miRNAs. (A) Scheme of MIR172b. Black boxes = exons, grey box = position of the pri-miRNA, thin line = introns. The arrows denote the position of the primers used in (B). (B) RNA from the AtGRP7-ox lines D and G and Col-2 wt was analysed by RT-PCR. The amplification products corresponding to the intron-retained form and the fully spliced forms are indicated by arrowheads and asterisks, respectively. PP2A served as a control. DNA = genomic DNA. (C) The ratio of intron-retained versus spliced pri-miR172b was quantified using Bioanalyzer DNA1000 chips. Shown is the mean of two reps. (D) Scheme of the non-protein-coding RNA harbouring MIR162a. Black boxes = exons, grey box = position of the pri-miRNA, open boxes = annotated 5′and 3′UTRs, thin line = introns. The arrows denote the position of the primers used in (E). (E) RNA from the AtGRP7-ox lines D and G and Col-2 wt was analysed by RT-PCR. The transcript forms corresponding to the amplification products are indicated. The rhombus denotes an alternative version of the 168 nt band generated by an alternative 3′splice site 3 nt downstream of the authentic 3′splice site. PP2A served as a control. DNA = genomic DNA. (F) The ratio of the alternative splice forms versus the spliced form was quantified using Bioanalyzer DNA1000 chips. Shown is the mean of two reps.
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Figure 6: AtGRP7 affects alternative splicing of pri-miRNAs. (A) Scheme of MIR172b. Black boxes = exons, grey box = position of the pri-miRNA, thin line = introns. The arrows denote the position of the primers used in (B). (B) RNA from the AtGRP7-ox lines D and G and Col-2 wt was analysed by RT-PCR. The amplification products corresponding to the intron-retained form and the fully spliced forms are indicated by arrowheads and asterisks, respectively. PP2A served as a control. DNA = genomic DNA. (C) The ratio of intron-retained versus spliced pri-miR172b was quantified using Bioanalyzer DNA1000 chips. Shown is the mean of two reps. (D) Scheme of the non-protein-coding RNA harbouring MIR162a. Black boxes = exons, grey box = position of the pri-miRNA, open boxes = annotated 5′and 3′UTRs, thin line = introns. The arrows denote the position of the primers used in (E). (E) RNA from the AtGRP7-ox lines D and G and Col-2 wt was analysed by RT-PCR. The transcript forms corresponding to the amplification products are indicated. The rhombus denotes an alternative version of the 168 nt band generated by an alternative 3′splice site 3 nt downstream of the authentic 3′splice site. PP2A served as a control. DNA = genomic DNA. (F) The ratio of the alternative splice forms versus the spliced form was quantified using Bioanalyzer DNA1000 chips. Shown is the mean of two reps.
Mentions: AtGRP7 overexpression leads to reduced levels of miR172b and miR172b* with a concomitant increase in pri-miR172b which contains introns both 5′ and 3′ of the stem–loop structure (Figure 6A). Because splicing of introns located 3′ of the stem–loop can impact accumulation of mature miRNAs (73,74), we asked whether AtGRP7 affects splicing of pri-miR172b. In wt plants, some pri-miR172b retaining the intron was detected in addition to the spliced form. In AtGRP7-ox plants, the spliced pri-miR172b accumulated at the expense of the intron retained form, indicating that AtGRP7 indeed affects splicing of pri-miRNAs (Figure 6B and C). In contrast, splicing of the introns located upstream of the hairpin was not altered (not shown). Thus, an enhanced assembly of the spliceosome at the 5′end of the downstream intron in AtGRP7-ox plants may interfere with pri-miRNA processing. Alternatively, a slower release of miR172b and miR172b* caused by AtGRP7 binding may allow more efficient assembly of the spliceosome.

Bottom Line: AtGRP7 overexpression caused a significant reduction in the level of 30 miRNAs and an increase for 14 miRNAs with a minimum log2 fold change of ± 0.5.Mutation of an arginine in the RNA recognition motif abrogated in vivo binding and the effect on miRNA and pri-miRNA levels, indicating that AtGRP7 inhibits processing of these pri-miRNAs by direct binding.Thus, AtGRP7 is an hnRNP-like protein with a role in processing of pri-miRNAs in addition to its role in pre-mRNA splicing.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Physiology, Bielefeld University.

Show MeSH