Alternative polyadenylation (APA) is a widespread mechanism for gene regulation and has been implicated in flowering, but the molecular basis governing the choice of a specific poly(A) site during the vegetative-to-reproductive growth transition remains unclear. Here we characterize HLP1, an hnRNP A/B protein as a novel regulator for pre-mRNA 3′-end processing in Arabidopsis. Genetic analysis reveals that HLP1 suppresses Flowering Locus C (FLC), a key repressor of flowering in Arabidopsis. Genome-wide mapping of HLP1-RNA interactions indicates that HLP1 binds preferentially to A-rich and U-rich elements around cleavage and polyadenylation sites, implicating its role in 3′-end formation. We show HLP1 is significantly enriched at transcripts involved in RNA metabolism and flowering. Comprehensive profiling of the poly(A) site usage reveals that HLP1 mutations cause thousands of poly(A) site shifts. A distal-to-proximal poly(A) site shift in the flowering regulator FCA, a direct target of HLP1, leads to upregulation of FLC and delayed flowering. Our results elucidate that HLP1 is a novel factor involved in 3′-end processing and controls reproductive timing via targeting APA.

Summary: Flowering is exquisitely regulated by both promotive and inhibitory factors. Molecular genetic studies with Arabidopsis have verified several epigenetic repressors that regulate flowering time. However, the roles of chromatin remodeling factors in developmental processes have not been well explored in Oryza sativa (rice). We identified a chromatin remodeling factor OsVIL2 (O. sativa VIN3‐LIKE 2) that promotes flowering. OsVIL2 contains a plant homeodomain (PHD) finger, which is a conserved motif of histone binding proteins. Insertion mutations in OsVIL2 caused late flowering under both long and short days. In osvil2 mutants OsLFL1 expression was increased, but that of Ehd1, Hd3a and RFT1 was reduced. We demonstrated that OsVIL2 is bound to native histone H3 in vitro. Chromatin immunoprecipitation analyses showed that OsVIL2 was directly associated with OsLFL1 chromatin. We also observed that H3K27me3 was significantly enriched by OsLFL1 chromatin in the wild type, but that this enrichment was diminished in the osvil2 mutants. These results indicated that OsVIL2 epigenetically represses OsLFL1 expression. We showed that OsVIL2 physically interacts with OsEMF2b, a component of polycomb repression complex 2. As observed from osvil2, a null mutation of OsEMF2b caused late flowering by increasing OsLFL1 expression and decreasing Ehd1 expression. Thus, we conclude that OsVIL2 functions together with PRC2 to induce flowering by repressing OsLFL1.

Significance: The functional relationship between protein arginine methyltransferases (PRMTs) and ribosome biogenesis was proposed a decade ago, but the underlying mechanism still remains elusive. In this work, we demonstrate for the first time to our knowledge that the coexistence of two pre-rRNA processing pathways is conserved in plants; and Arabidopsis PRMT3 is required for the balance between these pathways. These findings uncover an important link between PRMT and proper pre-rRNA processing, which not only extends our understanding of the regulatory scope of PRMTs but also reveals the complexity of regulation of ribosome biogenesis. The alternative pre-rRNA processing pathways in plants modulated by PRMT3 may be conserved in other multicellular organisms, thereby shedding light on PRMT functions and regulation of ribosome biogenesis in animals.

Ribosome biogenesis is crucial for plant growth and environmental acclimation. Processing of ribosomal RNAs (rRNAs) is an essential step in ribosome biogenesis and begins with transcription of the rDNA. The resulting precursor-rRNA (pre-rRNA) transcript undergoes systematic processing, where multiple endonucleolytic and exonucleolytic cleavages remove the external and internal transcribed spacers (ETS and ITS). The processing sites and pathways for pre-rRNA processing have been deciphered in Saccharomyces cerevisiae and, to some extent, in Xenopus laevis, mammalian cells, and Arabidopsis (Arabidopsis thaliana). However, the processing sites and pathways remain largely unknown in crops, particularly in monocots such as rice (Oryza sativa), one of the most important food resources in the world. Here, we identified the rRNA precursors produced during rRNA biogenesis and the critical endonucleolytic cleavage sites in the transcribed spacer regions of pre-rRNAs in rice. We further found that two pre-rRNA processing pathways, distinguished by the order of 5′ ETS removal and ITS1 cleavage, coexist in vivo. Moreover, exposing rice to chilling stress resulted in the inhibition of rRNA biogenesis mainly at the pre-rRNA processing level, suggesting that these energy-intensive processes may be reduced to increase acclimation and survival at lower temperatures. Overall, our study identified the pre-rRNA processing pathway in rice and showed that ribosome biogenesis is quickly inhibited by low temperatures, which may shed light on the link between ribosome biogenesis and environmental acclimation in crop plants.

Plant microRNAs (miRNAs) associate with ARGONAUTE1 (AGO1) to direct post-transcriptional gene silencing and regulate numerous biological processes. Although AGO1 predominantly binds miRNAs in vivo, it also associates with endogenous small interfering RNAs (siRNAs). It is unclear whether the miRNA/siRNA balance affects miRNA activities. Here we report that FIERY1 (FRY1), which is involved in 5′−3′ RNA degradation, regulates miRNA abundance and function by suppressing the biogenesis of ribosomal RNA-derived siRNAs (risiRNAs). In mutants of FRY1 and the nuclear 5′−3′ exonuclease genes XRN2 and XRN3, we find that a large number of 21-nt risiRNAs are generated through an endogenous siRNA biogenesis pathway. The production of risiRNAs correlates with pre-rRNA processing defects in these mutants. We also show that these risiRNAs are loaded into AGO1, causing reduced loading of miRNAs. This study reveals a previously unknown link between rRNA processing and miRNA accumulation.