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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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Pri-miRNA levels are elevated at the expense of mature miRNAs in AtGRP7-ox plants. The levels of the pri-miRNAs were analyzed in AtGRP7-ox, AtGRP7-R49Q-ox and C24 wt plants (A) and in the AtGRP7-ox lines D and G and Col-2 (B, C). Pri-miR398a, b and c, pri-miR390b, pri-miR172b, pri-miR159a and pri-miR399b correspond to miRNAs with reduced level in AtGRP7-ox plants. Pri-miR395e and pri-miR319b correspond to miRNAs with elevated levels in AtGRP7-ox plants, and pri-miR408a and pri-miR171c correspond to miRNAs that are not affected by AtGRP7 overexpression. Data are based on three biological reps. Asterisks denote a significant difference according to Student's t-test (P < 0.05).
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Figure 4: Pri-miRNA levels are elevated at the expense of mature miRNAs in AtGRP7-ox plants. The levels of the pri-miRNAs were analyzed in AtGRP7-ox, AtGRP7-R49Q-ox and C24 wt plants (A) and in the AtGRP7-ox lines D and G and Col-2 (B, C). Pri-miR398a, b and c, pri-miR390b, pri-miR172b, pri-miR159a and pri-miR399b correspond to miRNAs with reduced level in AtGRP7-ox plants. Pri-miR395e and pri-miR319b correspond to miRNAs with elevated levels in AtGRP7-ox plants, and pri-miR408a and pri-miR171c correspond to miRNAs that are not affected by AtGRP7 overexpression. Data are based on three biological reps. Asterisks denote a significant difference according to Student's t-test (P < 0.05).

Mentions: The altered steady-state level of miRNAs in AtGRP7-ox plants could be a consequence of altered levels of pri-miRNAs or altered fates of mature miRNAs. RT-PCR analysis revealed significantly elevated levels of pri-miR398a, pri-miR398b and pri-miR398c in AtGRP7-ox but not AtGRP7-R49Q-ox plants (Figure 4A and B). Similarly, the primary transcript for miR390b that initiates TAS3 cleavage in ta-siRNA generation was significantly elevated in AtGRP7-ox but not AtGRP7-R49Q-ox plants. Furthermore, we found elevated levels of pri-miR172b and pri-miR159a in independent AtGRP7-ox lines (Figure 4B). As the miR159 precursor is processed by an unusual mechanism with the initial cut near the loop of the stem rather than at its base (64), AtGRP7 appears to affect miRNA precursors processed by the conventional base-to-loop mechanism and precursors processed in loop-to-base direction (Supplementary Tables S4 and S5). The elevated pri-miR399b levels (Figure 4B) correlate with the reduced miR399b level found by RNA-seq.


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)

Pri-miRNA levels are elevated at the expense of mature miRNAs in AtGRP7-ox plants. The levels of the pri-miRNAs were analyzed in AtGRP7-ox, AtGRP7-R49Q-ox and C24 wt plants (A) and in the AtGRP7-ox lines D and G and Col-2 (B, C). Pri-miR398a, b and c, pri-miR390b, pri-miR172b, pri-miR159a and pri-miR399b correspond to miRNAs with reduced level in AtGRP7-ox plants. Pri-miR395e and pri-miR319b correspond to miRNAs with elevated levels in AtGRP7-ox plants, and pri-miR408a and pri-miR171c correspond to miRNAs that are not affected by AtGRP7 overexpression. Data are based on three biological reps. Asterisks denote a significant difference according to Student's t-test (P < 0.05).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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Figure 4: Pri-miRNA levels are elevated at the expense of mature miRNAs in AtGRP7-ox plants. The levels of the pri-miRNAs were analyzed in AtGRP7-ox, AtGRP7-R49Q-ox and C24 wt plants (A) and in the AtGRP7-ox lines D and G and Col-2 (B, C). Pri-miR398a, b and c, pri-miR390b, pri-miR172b, pri-miR159a and pri-miR399b correspond to miRNAs with reduced level in AtGRP7-ox plants. Pri-miR395e and pri-miR319b correspond to miRNAs with elevated levels in AtGRP7-ox plants, and pri-miR408a and pri-miR171c correspond to miRNAs that are not affected by AtGRP7 overexpression. Data are based on three biological reps. Asterisks denote a significant difference according to Student's t-test (P < 0.05).
Mentions: The altered steady-state level of miRNAs in AtGRP7-ox plants could be a consequence of altered levels of pri-miRNAs or altered fates of mature miRNAs. RT-PCR analysis revealed significantly elevated levels of pri-miR398a, pri-miR398b and pri-miR398c in AtGRP7-ox but not AtGRP7-R49Q-ox plants (Figure 4A and B). Similarly, the primary transcript for miR390b that initiates TAS3 cleavage in ta-siRNA generation was significantly elevated in AtGRP7-ox but not AtGRP7-R49Q-ox plants. Furthermore, we found elevated levels of pri-miR172b and pri-miR159a in independent AtGRP7-ox lines (Figure 4B). As the miR159 precursor is processed by an unusual mechanism with the initial cut near the loop of the stem rather than at its base (64), AtGRP7 appears to affect miRNA precursors processed by the conventional base-to-loop mechanism and precursors processed in loop-to-base direction (Supplementary Tables S4 and S5). The elevated pri-miR399b levels (Figure 4B) correlate with the reduced miR399b level found by RNA-seq.

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