Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs

Abstract

Arabidopsis thaliana HYL1 is a nuclear double-stranded RNA-binding protein involved in the maturation of pri-miRNAs. A quantitative real-time PCR platform for parallel quantification of 176 pri-miRNAs was used to reveal strong accumulation of 57 miRNA precursors in the hyl1 mutant that completely lacks HYL1 protein. This approach enabled us for the first time to pinpoint particular members of MIRNA family genes that require HYL1 activity for efficient maturation of their precursors. Moreover, the accumulation of miRNA precursors in the hyl1 mutant gave us the opportunity to carry out 3′ and 5′ RACE experiments which revealed that some of these precursors are of unexpected length. The alignment of HYL1-dependent miRNA precursors to A. thaliana genomic sequences indicated the presence of introns in 12 out of 20 genes studied. Some of the characterized intron-containing pri-miRNAs undergo alternative splicing such as exon skipping or usage of alternative 5′ splice sites suggesting that this process plays a role in the regulation of miRNA biogenesis. In the hyl1 mutant intron-containing pri-miRNAs accumulate alongside spliced pri-miRNAs suggesting the recruitment of HYL1 into the miRNA precursor maturation pathway before their splicing occurs.

RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii

“…In the A. thaliana HYL1 mutant background, some pri-miRNAs of unusual length, containing introns, accumulate (56). In Chlamydomonas, the efficiency of splicing of intronic pri-miRNAs was apparently unchanged in the dus16-1 background.…”

The role of decapping proteins in the miRNA accumulation inArabidopsis thaliana

“…pri-miRNAs are initially transcribed by RNA polymerase II and then processed into mature miRNAs by Dicer-like 1 (DCL1) together with HYPONASTIC LEAVES 1 (HYL1) and SERRATE (SE) in A. thaliana. [13][14][15][16][17] Mature miRNAs are methylated by HEN1 and mainly incorporated into Argonaute 1 (AGO1) to regulate gene expression on the mRNAs in a sequence specific manner. [18][19][20][21] dcl1-and ago1-null mutants show lethal phenotypes, indicating that miRNAs participate in plant development.…”

The crosstalk between plant microRNA biogenesis factors and the spliceosome

“…Our work showed that pri-miRNAs of plants can have extremely complex structures and often contain multiple introns. 40 We anticipate that other not-yet-identified features and mechanisms contribute to the tight regulation of miRNA levels in plants.…”

“…Surprisingly, for the miRNA genes representing independent transcriptional units with known structures, it turned out that they are very long (from 378 bp to 4975 bp), and many of them contain multiple introns. 17,18,39,40 These introns undergo complex alternative splicing, providing a possible mechanism for the regulation of miRNA processing in A. thaliana. 38,41 Our estimations have shown that approximately 50% of A. thaliana miRNA genes contains intron(s).…”

“…43 Pri-miRNA polyadenylation site selection depends on the functional 5′ splice site Studies on plant pri-miRNA structures revealed that multiple polyadenylation sites might co-exist. 40,49,50 In the case of Arabidopsis pri-miR163, 2 polyA sites were identified: the proximal site that is located within the intron, and the distal site which is located at the end of the gene. 42,43 In the wild type A. thaliana cells, 60% of pri-miR163 is terminated at the distal site.…”