Proteins that specifically bind double-stranded RNA (dsRNA) are involved in the regulation of cellular signaling events and gene expression, and are characterized by a conserved dsRNA-binding motif (dsRBM). Here we report the biochemical properties of nine such gene products, each containing one or two dsRBMs: four Arabidopsis Dicer-like proteins (DCL1-4), Arabidopsis HYL1 and four of its homologs (DRB2, DRB4, DRB5 and OsDRB1). DCL1, DCL3, HYL1 and the four HYL1 homologs exhibit significant dsRNA-binding activity, indicating that these proteins are involved in RNA metabolism. The dsRBMs from dsRBM-containing proteins (dsRBPs) also function as a protein-protein interaction domain and homo- and heterodimerization are essential for biological functioning of these proteins. We show that DRB4 interacts specifically with DCL4, and HYL1 most strongly interacts with DCL1. These results indicate that each HYL1/DRB family protein interacts with one specific partner among the four Dicer-like proteins. Localization studies using GFP fusion proteins demonstrate that DCL1, DCL4, HYL1 and DRB4 localize in the nucleus, while DRB2 is present in the cytoplasm. Subcellular localizations of HYL1, DRB4, DCL1 and DCL4 further strengthen the notion that HYL1 and DCL1, and DRB4 and DCL4, exist as complexes. The presented data suggest that each member of the HYL1/DRB protein family may individually modulate Dicer function through heterodimerization with a Dicer-like protein in vivo.

Transcription and mRNA processing are regulated by phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II, which consists of tandem repeats of a Y 1 S 2 P 3 T 4 S 5 P 6 S 7 heptapeptide. Previous studies showed that members of the plant CTD phosphatase-like (CPL) protein family differentially regulate osmotic stress-responsive and abscisic acidresponsive transcription in Arabidopsis thaliana. Here we report that AtCPL1 and AtCPL2 specifically dephosphorylate Ser-5 of the CTD heptad in Arabidopsis RNA polymerase II, but not Ser-2. An N-terminal catalytic domain of CPL1, which suffices for CTD Ser-5 phosphatase activity in vitro, includes a signature DXDXT acylphosphatase motif, but lacks a breast cancer 1 CTD, which is an essential component of the fungal and metazoan Fcp1 CTD phosphatase enzymes. The CTD of CPL1, which contains two putative doublestranded RNA binding motifs, is essential for the in vivo function of CPL1 and includes a C-terminal 23-aa signal responsible for its nuclear targeting. CPL2 has a similar domain structure but contains only one double-stranded RNA binding motif. Combining mutant alleles of CPL1 and CPL2 causes synthetic lethality of the male but not the female gametes. These results indicate that CPL1 and CPL2 exemplify a unique family of CTD Ser-5-specific phosphatases with an essential role in plant growth and development.T ranscriptional induction of genes that encode stress tolerance determinants is an integral part of the survival strategy of plants in adverse environments. The Arabidopsis thaliana responsive to dehydration (RD) genes are prototypal outputs of stress signal integration activated by low temperature, hyperosmolarity, and the plant hormone abscisic acid (ABA). The stress-inducible promoter of the RD29a gene contains dehydration͞cold-responsive elements and ABA-responsive elements that are the targets of distinct families of DNA binding transcription factors (1, 2). The plant stress response is also regulated by proteins that impact the core RNA polymerase II (Pol II) transcriptional machinery, the mRNA maturation process, and chromatin structure (3-9). Analysis of Arabidopsis mutants that display hyperinduction of RD29a expression under stress conditions have identified a family of C-terminal domain (CTD) phosphatase-like (CPL) genes that negatively regulate stressresponsive transcription (5, 6). The CPL1 and CPL3 genes discovered in the screen for hyperinduction are so named because they encode large polypeptides (967 and 1,241 aa, respectively) with local primary structure similarity to the Fcp1 family of fungal and metazoan protein serine phosphatases, which regulate transcription by dephosphorylating the CTD of the largest subunit of RNA Pol II (10).The Pol II CTD is composed of a tandemly repeated heptapeptide of consensus sequence Y . The number of CTD heptad repeats varies widely among species and correlates roughly with evolutionary complexity; e.g., mammals have 52 repeats, Drosophila has 42 repeats, fission yeast Schizosacc...

Arabidopsis thaliana encodes four Dicer-like (DCL) proteins and five dsRNA-binding (DRB) proteins. We have previously demonstrated that DCL4 specifically interacts with DRB4 in vitro. Here we describe the interaction between DCL4 and DRB4 in vivo. The phenotype of a mutant with a defect in DCL4 (dcl4-2) was similar to that of a mutant with a defect in DRB4 (drb4-1): both mutant plants had elongated and downwardly curled rosette leaves and over-accumulated anthocyanin. In immunoprecipitation experiments with either anti-DCL4 or anti-DRB4 antibody and crude extracts of wild-type Arabidopsis plants, co-immunoprecipitation of DCL4 and DRB4 was detected, indicating that DCL4 interacts with DRB4 in vivo. This interaction was confirmed by immunoprecipitation experiments using extracts from dcl4-2, drb4-1, or transgenic plants expressing the hemagglutinin-tagged version of DCL4 or DRB4. The results of immunoprecipitation experiments also suggest that most DCL4 is associated with DRB4, but that some DRB4 is free or associated with other proteins. Reduced accumulation of the TAS1 and TAS3 trans-acting siRNA (ta-siRNA) and over accumulation of their target mRNAs (At5g18040 and auxin response factors ARF3 and ARF4) were detected in both drb4-1 and dcl4-2 mutants. These results indicate that DRB4, together with DCL4, functions in the ta-siRNA biogenesis.

In Arabidopsis thaliana, Dicer-like 3 (DCL3) and Dicer-like 4 (DCL4) cleave long, perfect double-stranded RNAs (dsRNAs) into 24 and 21 nucleotides (nt) small interfering RNAs, respectively, which in turn function in RNA-directed DNA methylation and RNA interference, respectively. To reveal how DCL3 and DCL4 individually recognize long perfect dsRNAs as substrates, we biochemically characterized DCL3 and DCL4 and compared their enzymatic properties. DCL3 preferentially cleaves short dsRNAs with 5′ phosphorylated adenosine or uridine and a 1 nt 3′ overhang, whereas DCL4 cleaves long dsRNAs with blunt ends or with a 1 or 2 nt 3′ overhang with similar efficiency. DCL3 produces 24 nt RNA duplexes with 2 nt 3′ overhangs by the 5′ counting rule. Inorganic phosphate, NaCl and KCl enhance DCL3 activity but inhibit DCL4 activity. These results indicate that plants use DCLs with distinct catalytic profiles to ensure each dsRNA substrate generates only a specific length of siRNAs that trigger a unique siRNA-mediated response.