Gordon G. Simpson

Autoregulation of FCA pre-mRNA processing controls Arabidopsis flowering time

The timing of the transition to flowering is critical for reproductive success in plants. Arabidopsis FCA encodes an RNA‐binding protein that promotes flowering. FCA expression is regulated through alternative processing of its pre‐mRNA. We demonstrate here that FCA negatively regulates its own expression by ultimately promoting cleavage and polyadenylation within intron 3. This causes the production of a truncated, inactive transcript at the expense of the full‐length FCA mRNA, thus limiting the expression of active FCA protein. We show that this negative autoregulation is under developmental control and requires the FCA WW protein interaction domain. Removal of introns from FCA bypasses the autoregulation, and the resulting increased levels of FCA protein overcomes the repression of flowering normally conferred through the up‐regulation of FLC by active FRI alleles. The negative autoregulation of FCA may therefore have evolved to limit FCA activity and hence control flowering time.

Functional Significance of the Alternative Transcript Processing of the Arabidopsis Floral Promoter FCA

The Arabidopsis gene FCA encodes an RNA binding protein that functions to promote the floral transition. The FCA transcript is alternatively processed to yield four transcripts, the most abundant of which is polyadenylated within intron 3. We have analyzed the role of the alternative processing on the floral transition. The introduction of FCA intronless transgenes resulted in increased FCA protein levels and accelerated flowering, but no role in flowering was found for products of the shorter transcripts. The consequences of the alternative processing on the FCA expression pattern were determined using a series of translational FCA–β-glucuronidase fusions. The inclusion of FCA genomic sequence containing the alternatively processed intron 3 restricted the expression of the transgene predominantly to shoot and root apices and young flower buds. Expression of this fusion also was delayed developmentally. Therefore, the alternative processing of the FCA transcript limits, both spatially and temporally, the amount of functional FCA protein. Expression in roots prompted an analysis of root development, which indicated that FCA functions more generally than in the control of the floral transition.

Caffeoyl Shikimate Esterase (CSE) Is an Enzyme in the Lignin Biosynthetic Pathway in Arabidopsis

Lignin Biosynthesis Complications Lignin is a polymer that lends its sturdy properties to wood and makes plant cell walls tougher, which creates problems for chemists converting cellulosic plant biomass into biofuels. Vanholme et al. (p. 1103, published online 15 August; see the cover) have identified a new step in the biosynthetic pathway of lignin in Arabidopsis in which caffeoyl shikimate esterase catalyzes synthesis of caffeate. Cellulose from mutant plants, which had reduced amounts of lignin, was more efficiently processed into glucose. A key enzyme involved in lignin biosynthesis is identified and characterized in the model plant Arabidopsis. Lignin is a major component of plant secondary cell walls. Here we describe caffeoyl shikimate esterase (CSE) as an enzyme central to the lignin biosynthetic pathway. Arabidopsis thaliana cse mutants deposit less lignin than do wild-type plants, and the remaining lignin is enriched in p-hydroxyphenyl units. Phenolic metabolite profiling identified accumulation of the lignin pathway intermediate caffeoyl shikimate in cse mutants as compared to caffeoyl shikimate levels in the wild type, suggesting caffeoyl shikimate as a substrate for CSE. Accordingly, recombinant CSE hydrolyzed caffeoyl shikimate into caffeate. Associated with the changes in lignin, the conversion of cellulose to glucose in cse mutants increased up to fourfold as compared to that in the wild type upon saccharification without pretreatment. Collectively, these data necessitate the revision of currently accepted models of the lignin biosynthetic pathway.

How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?

RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%-40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools.

Environmental-Dependent Acceleration of a Developmental Switch: The Floral Transition

The transition from vegetative growth to reproductive growth in plants in which flowers are produced requires the activation of specific genes. Simpson and Dean discuss two recent reports that characterize the FLOWERING LOCUS T ( FT ) gene in Arabidopsis , which is part of the floral transition pathway. Unlike many of the known genes that initiate flower production, the FT gene appears to encode a membrane-associated protein that could function in signaling from the cell surface.

UBP1, a novel hnRNP‐like protein that functions at multiple steps of higher plant nuclear pre‐mRNA maturation

Efficient splicing of higher plant pre‐mRNAs depends on AU‐ or U‐rich sequences in introns. Moreover, AU‐rich sequences present in 3′‐untranslated regions (3′‐UTRs) may play a role in 3′ end processing of plant mRNAs. Here, we describe the cloning and characterization of a Nicotiana plumbaginifolia nuclear protein that can be cross‐linked to U‐rich intron and 3′‐UTR sequences in vitro, and associates with nuclear poly(A)+ RNA in vivo. The protein, UBP1, strongly enhances the splicing of otherwise inefficiently processed introns when overexpressed in protoplasts. It also increases the accumulation of reporter mRNAs that contain suboptimal introns or are intronless. The enhanced accumulation is apparently due to UBP1 interacting with the 3′‐UTR and protecting mRNA from exonucleolytic degradation. The effect on mRNA accumulation but not on mRNA splicing was found to be promoter specific. The fact that these effects of UBP1 can be separated suggests that they represent two independent activities. The properties of UBP1 indicate that it is an hnRNP protein that functions at multiple steps to facilitate the nuclear maturation of plant pre‐mRNAs.

Direct sequencing of Arabidopsis thaliana RNA reveals patterns of cleavage and polyadenylation

It has recently been shown that RNA 3′-end formation plays a more widespread role in controlling gene expression than previously thought. To examine the impact of regulated 3′-end formation genome-wide, we applied direct RNA sequencing to A. thaliana. Here we show the authentic transcriptome in unprecedented detail and describe the effects of 3′-end formation on genome organization. We reveal extreme heterogeneity in RNA 3′ ends, discover previously unrecognized noncoding RNAs and propose widespread reannotation of the genome. We explain the origin of most poly(A)+ antisense RNAs and identify cis elements that control 3′-end formation in different registers. These findings are essential to understanding what the genome actually encodes, how it is organized and how regulated 3′-end formation affects these processes.

An allelic series reveals essential roles for FY in plant development in addition to flowering-time control

The autonomous pathway functions to promote flowering in Arabidopsis by limiting the accumulation of the floral repressor FLOWERING LOCUS C (FLC). Within this pathway FCA is a plant-specific, nuclear RNA-binding protein, which interacts with FY, a highly conserved eukaryotic polyadenylation factor. FCA and FY function to control polyadenylation site choice during processing of the FCA transcript. Null mutations in the yeast FY homologue Pfs2p are lethal. This raises the question as to whether these essential RNA processing functions are conserved in plants. Characterisation of an allelic series of fy mutations reveals that null alleles are embryo lethal. Furthermore, silencing of FY, but not FCA, is deleterious to growth in Nicotiana. The late-flowering fy alleles are hypomorphic and indicate a requirement for both intact FY WD repeats and the C-terminal domain in repression of FLC. The FY C-terminal domain binds FCA and in vitro assays demonstrate a requirement for both C-terminal FY-PPLPP repeats during this interaction. The expression domain of FY supports its roles in essential and flowering-time functions. Hence, FY may mediate both regulated and constitutive RNA 3′-end processing.

Transcription termination and chimeric RNA formation controlled by Arabidopsis thaliana FPA.

Alternative cleavage and polyadenylation influence the coding and regulatory potential of mRNAs and where transcription termination occurs. Although widespread, few regulators of this process are known. The Arabidopsis thaliana protein FPA is a rare example of a trans-acting regulator of poly(A) site choice. Analysing fpa mutants therefore provides an opportunity to reveal generic consequences of disrupting this process. We used direct RNA sequencing to quantify shifts in RNA 3′ formation in fpa mutants. Here we show that specific chimeric RNAs formed between the exons of otherwise separate genes are a striking consequence of loss of FPA function. We define intergenic read-through transcripts resulting from defective RNA 3′ end formation in fpa mutants and detail cryptic splicing and antisense transcription associated with these read-through RNAs. We identify alternative polyadenylation within introns that is sensitive to FPA and show FPA-dependent shifts in IBM1 poly(A) site selection that differ from those recently defined in mutants defective in intragenic heterochromatin and DNA methylation. Finally, we show that defective termination at specific loci in fpa mutants is shared with dicer-like 1 (dcl1) or dcl4 mutants, leading us to develop alternative explanations for some silencing roles of these proteins. We relate our findings to the impact that altered patterns of 3′ end formation can have on gene and genome organisation.

Noncanonical Translation Initiation of the Arabidopsis Flowering Time and Alternative Polyadenylation Regulator FCA

This study shows that translation of the flowering time regulator FCA initiates at a CUG, rather than at an AUG, codon and that specific sequences within the 5′ untranslated region are required for this noncanonical translation initiation. Our bioinformatic analyses suggest that there are at least 10 other Arabidopsis genes that initiate translation at CUG codons. The RNA binding protein FCA regulates the floral transition and is required for silencing RNAs corresponding to specific noncoding sequences in the Arabidopsis thaliana genome. Through interaction with the canonical RNA 3′ processing machinery, FCA affects alternative polyadenylation of many transcripts, including antisense RNAs at the locus encoding the floral repressor FLC. This potential for widespread alteration of gene regulation clearly needs to be tightly regulated, and we have previously shown that FCA expression is autoregulated through poly(A) site choice. Here, we show distinct layers of FCA regulation that involve sequences within the 5′ region that regulate noncanonical translation initiation and alter the expression profile. FCA translation in vivo occurs exclusively at a noncanonical CUG codon upstream of the first in-frame AUG. We fully define the upstream flanking sequences essential for its selection, revealing features that distinguish this from other non-AUG start site mechanisms. Bioinformatic analysis identified 10 additional Arabidopsis genes that likely initiate translation at a CUG codon. Our findings reveal further unexpected complexity in the regulation of FCA expression with implications for its roles in regulating flowering time and gene expression and more generally show plant mRNA exceptions to AUG translation initiation.