Noncoding RNA is emerging as an important regulator of gene expression in many organisms. We are characterizing RNA-mediated chromatin silencing of the Arabidopsis major floral repressor gene, FLC. Through suppressor mutagenesis, we identify a requirement for CstF64 and CstF77, two conserved RNA 3'-end-processing factors, in FLC silencing. However, FLC sense transcript 3' processing is not affected in the mutants. Instead, CstF64 and CstF77 are required for 3' processing of FLC antisense transcripts. A specific RNA-binding protein directs their activity to a proximal antisense polyadenylation site. This targeted processing triggers localized histone demethylase activity and results in reduced FLC sense transcription. Targeted 3' processing of antisense transcripts may be a common mechanism triggering transcriptional silencing of the corresponding sense gene.

The Arabidopsis GNOM gene encodes an ARF GDP/GTP exchange factor involved in embryonic axis formation and polar localisation of the auxin efflux regulator PIN1. To examine whether GNOM also plays a role in post-embryonic development and to clarify its involvement in auxin transport,we have characterised newly isolated weak gnom alleles as well as trans-heterozygotes of complementing strong alleles. These genotypes form a phenotypic series of GNOM activity in post-embryonic development,with auxin-related defects, especially in the maintenance of primary root meristem activity and in the initiation and organisation of lateral root primordia. Our results suggest a model for GNOM action mediating auxin transport in both embryogenesis and post-embryonic organ development.

SummaryAntisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just downstream of the major sense transcript poly(A) site and terminates either early or extends into the FLC promoter region. We now show that splicing of COOLAIR is functionally important. This was revealed through analysis of a hypomorphic mutation in the core spliceosome component PRP8. The prp8 mutation perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms.

In animals, cyclin-dependent kinase inhibitors (CKIs) are important regulators of cell cycle progression. Recently, putative CKIs were also identified in plants, and in previous studies, Arabidopsis thaliana plants misexpressing CKIs were found to have reduced endoreplication levels and decreased numbers of cells consistent with a function of CKIs in blocking the G1-S cell cycle transition. Here, we demonstrate that at least one inhibitor from Arabidopsis, ICK1/KRP1, can also block entry into mitosis but allows S-phase progression causing endoreplication. Our data suggest that plant CKIs act in a concentrationdependent manner and have an important function in cell proliferation as well as in cell cycle exit and in turning from a mitotic to an endoreplicating cell cycle mode. Endoreplication is usually associated with terminal differentiation; we observed, however, that cell fate specification proceeded independently from ICK1/KRP1-induced endoreplication. Strikingly, we found that endoreplicated cells were able to reenter mitosis, emphasizing the high degree of flexibility of plant cells during development. Moreover, we show that in contrast with animal CDK inhibitors, ICK1/KRP1 can move between cells. On the one hand, this challenges plant cell cycle control with keeping CKIs locally controlled, and on the other hand this provides a possibility of linking cell cycle control in single cells with the supracellular organization of a tissue or an organ.

Most DNA in the genomes of higher organisms does not encode proteins, yet much is transcribed by RNA polymerase II (RNAPII) into long non-coding RNAs (lncRNAs). The biological significance of most lncRNAs is largely unclear. Here, we identify a lncRNA (SVALKA) in a cold-sensitive region of the Arabidopsis genome. Mutations in SVALKA affect CBF1 expression and freezing tolerance. RNAPII read-through transcription of SVALKA results in a cryptic lncRNA overlapping CBF1 on the antisense strand, termed asCBF1. Our molecular dissection reveals that CBF1 is suppressed by RNAPII collision stemming from the SVALKA-asCBF1 lncRNA cascade. The SVALKA-asCBF1 cascade provides a mechanism to tightly control CBF1 expression and timing that could be exploited to maximize freezing tolerance with mitigated fitness costs. Our results provide a compelling example of local gene regulation by lncRNA transcription having a profound impact on the ability of plants to appropriately acclimate to challenging environmental conditions.

SUMMARY The essential helicase-like protein Sen1 mediates termination of RNA Polymerase II (Pol II) transcription at snoRNAs and other non-coding RNAs in yeast. A mutation in the Pol II subunit Rpb1 that increases the elongation rate increases readthrough transcription at Sen1-mediated terminators. Termination and growth defects in sen1 mutant cells are partially suppressed by a slowly transcribing Pol II mutant and are exacerbated by a faster transcribing Pol II mutant. Deletion of the nuclear exosome subunit Rrp6 allows visualization of non-coding RNA intermediates that are terminated but not yet processed. Sen1 mutants or faster transcribing Pol II increase the average lengths of pre-processed snoRNA, CUT, and SUT transcripts, while slowed Pol II transcription produces shorter transcripts. These connections between transcription rate and Sen1 activity support a model whereby kinetic competition between elongating Pol II and Sen1 helicase establishes the temporal and spatial window for early Pol II termination.

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In addition to their annotated transcript, many eukaryotic mRNA promoters produce divergent noncoding transcripts. To define determinants of divergent promoter directionality we used genomic replacement experiments. Sequences within non-coding transcripts specified their degradation pathways, and functional protein-coding transcripts could be produced in the divergent direction. To screen for mutants affecting the ratio of transcription in each direction, a bi-directional fluorescent protein reporter construct was introduced into the yeast non-essential gene deletion collection. We identified chromatin assembly as an important regulator of divergent transcription. Mutations in the CAF-I complex caused genome-wide de-repression of nascent divergent non-coding transcription. In opposition to the CAF-I chromatin assembly pathway, H3K56 hyperacetylation together with the nucleosome remodeler SWI/SNF facilitated divergent transcription by promoting rapid nucleosome turnover. We propose that these chromatin-mediated effects control divergent transcription initiation, complementing downstream pathways linked to early termination and degradation of the non-coding RNAs.