Brenda J. Reinhart

The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans.

The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events. Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated. Here we show that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3′ untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly controlled by let-7. A reporter gene bearing the lin-41 3′ untranslated region is temporally regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates lin-14 and lin-28 through RNA–RNA interactions with their 3′ untranslated regions. We propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing.

Prediction of Plant MicroRNA Targets

We predict regulatory targets for 14 Arabidopsis microRNAs (miRNAs) by identifying mRNAs with near complementarity. Complementary sites within predicted targets are conserved in rice. Of the 49 predicted targets, 34 are members of transcription factor gene families involved in developmental patterning or cell differentiation. The near-perfect complementarity between plant miRNAs and their targets suggests that many plant miRNAs act similarly to small interfering RNAs and direct mRNA cleavage. The targeting of developmental transcription factors suggests that many plant miRNAs function during cellular differentiation to clear key regulatory transcripts from daughter cell lineages.

MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5¿ region

MicroRNAs (miRNAs) are ∼22‐nucleotide noncoding RNAs that can regulate gene expression by directing mRNA degradation or inhibiting productive translation. Dominant mutations in PHABULOSA (PHB) and PHAVOLUTA (PHV) map to a miR165/166 complementary site and impair miRNA‐guided cleavage of these mRNAs in vitro. Here, we confirm that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb‐d mutants. In planta, disrupting miRNA pairing near the center of the miRNA complementary site had far milder developmental consequences than more distal mismatches. These differences correlated with differences in miRNA‐directed cleavage efficiency in vitro, where mismatch scanning revealed more tolerance for mismatches at the center and 3′ end of the miRNA compared to mismatches to the miRNA 5′ region. In this respect, miR165/166 resembles animal miRNAs in its pairing requirements. Pairing to the 5′ portion of the small silencing RNA appears crucial regardless of the mode of post‐transcriptional repression or whether it occurs in plants or animals, supporting a model in which this region of the silencing RNA nucleates pairing to its target.

Computational and Experimental Identification of C. elegans microRNAs.

MicroRNAs (miRNAs) constitute an extensive class of noncoding RNAs that are thought to regulate the expression of target genes via complementary base-pair interactions. To date, cloning has identified over 200 miRNAs from diverse eukaryotic organisms. Despite their success, such biochemical approaches are skewed toward identifying abundant miRNAs, unlike genome-wide, sequence-based computational predictions. We developed informatic methods to predict miRNAs in the C. elegans genome using sequence conservation and structural similarity to known miRNAs and generated 214 candidates. We confirmed the expression of four new miRNAs by Northern blotting and used a more sensitive PCR approach to verify the expression of ten additional candidates. Based on hypotheses underlying our computational methods, we estimate that the C. elegans genome may encode between 140 and 300 miRNAs and potentially many more.

A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco

Two major classes of small noncoding RNAs have emerged as important regulators of gene expression in eukaryotes, the short interfering RNAs (siRNAs) associated with RNA silencing and endogenous micro-RNAs (miRNAs) implicated in regulation of gene expression. Helper component-proteinase (HC-Pro) is a viral protein that blocks RNA silencing in plants. Here we examine the effect of HC-Pro on the accumulation of siRNAs and endogenous miRNAs. siRNAs were analyzed in transgenic tobacco plants silenced in response to three different classes of transgenes: sense-transgenes, inverted-repeat transgenes, and amplicon-transgenes. HC-Pro suppressed silencing in each line, blocking accumulation of the associated siRNAs and allowing accumulation of transcripts from the previously silenced loci. HC-Pro-suppression of silencing in the inverted-repeat- and amplicon-transgenic lines was accompanied by the apparent accumulation of long double-stranded RNAs and proportional amounts of small RNAs that are larger than the siRNAs that accumulate during silencing. Analysis of these results suggests that HC-Pro interferes with silencing either by inhibiting siRNA processing from double-stranded RNA precursors or by destabilizing siRNAs. In contrast to siRNAs, the accumulation of endogenous miRNAs was greatly enhanced in all of the HC-Pro-expressing lines. Thus, our results demonstrate that accumulation of siRNAs and miRNAs in plants can be differentially regulated by a viral protein. The fact that HC-Pro affects the miRNA pathway raises the possibility that this pathway is targeted by plant viruses as a means to control gene expression in the host.