Proper execution of transcriptional programs is a key requirement of gene expression regulation, demanding accurate control of timing and amplitude. How precisely the transcription machinery fulfills this task is not known. Using an in situ hybridization approach that detects single mRNA molecules, we measured mRNA abundance and transcriptional activity within single Saccharomyces cerevisiae cells. We found that expression levels for particular genes are higher than initially reported and can vary substantially among cells. However, variability for most constitutively expressed genes is unexpectedly small. Combining single-transcript measurements with computational modeling indicates that low expression variation is achieved by transcribing genes using single transcription-initiation events that are clearly separated in time, rather than by transcriptional bursts. In contrast, PDR5, a gene regulated by the transcription coactivator complex SAGA, is expressed using transcription bursts, resulting in larger variation. These data directly demonstrate the existence of multiple expression modes used to modulate the transcriptome.

Cellular messenger RNA levels are achieved by the combinatorial complexity of factors controlling transcription, yet the small number of molecules involved in these pathways fluctuates stochastically. It has not yet been experimentally possible to observe the activity of single polymerases on an endogenous gene to elucidate how these events occur in vivo. Here, we describe a method of fluctuation analysis of fluorescently labeled RNA to measure dynamics of nascent RNA—including initiation, elongation, and termination—at an active yeast locus. We find no transcriptional memory between initiation events, and elongation speed can vary by threefold throughout the cell cycle. By measuring the abundance and intranuclear mobility of an upstream transcription factor, we observe that the gene firing rate is directly determined by trans-activating factor search times.

Localization of beta-actin messenger RNA to sites of active actin polymerization modulates cell migration during embryogenesis, differentiation and possibly carcinogenesis. This localization requires the oncofetal protein ZBP1 (Zipcode binding protein 1), which binds to a conserved 54-nucleotide element in the 3'-untranslated region of the beta-actin mRNA known as the 'zipcode'. ZBP1 promotes translocation of the beta-actin transcript to actin-rich protrusions in primary fibroblasts and neurons. It is not known how the ZBP1-RNA complex achieves asymmetric protein sorting by localizing beta-actin mRNA. Here we show that chicken ZBP1 modulates the translation of beta-actin mRNA. ZBP1 associates with the beta-actin transcript in the nucleus and prevents premature translation in the cytoplasm by blocking translation initiation. Translation only occurs when the ZBP1-RNA complex reaches its destination at the periphery of the cell. At the endpoint of mRNA transport, the protein kinase Src promotes translation by phosphorylating a key tyrosine residue in ZBP1 that is required for binding to RNA. These sequential events provide both temporal and spatial control over beta-actin mRNA translation, which is important for cell migration and neurite outgrowth.

Yra1p/REF participates in mRNA export by recruiting the export receptor Mex67p to messenger ribonucleoprotein (mRNP) complexes. Yra1p also binds Sub2p, a DEAD box ATPase/RNA helicase implicated in splicing and required for mRNA export. We identified genetic and physical interactions between Yra1p, Sub2p, and Hpr1p, a protein involved in transcription elongation whose deletion leads to poly(A) ؉ RNA accumulation in the nucleus. By chromatin immunoprecipitation (ChIP) experiments, we show that Hpr1p, Sub2p, and Yra1p become associated with active genes during transcription elongation and that Hpr1p is required for the efficient recruitment of Sub2p and Yra1p. The data indicate that transcription and export are functionally linked and that mRNA export defects may be due in part to inefficient loading of essential mRNA export factors on the growing mRNP. We also identified functional interactions between Yra1p and the exosome components Rrp45p and Rrp6p. We show that yra1, sub2, and ⌬hpr1 mutants all present defects in mRNA accumulation and that deletion of RRP6 in yra1 mutants restores normal mRNA levels. The data support the hypothesis that an exosome-dependent surveillance mechanism targets improperly assembled mRNPs for degradation. mRNAs are exported from the nucleus as messenger ribonucleoprotein (mRNP) complexes which begin to be assembled during transcription. mRNA biogenesis requires multiple processing steps, including the addition of a 5Ј cap, splicing, and 3Ј-end formation, which have to be completed before the mRNA can be exported. Fully mature mRNPs are subsequently recognized by the essential mRNA export receptor Mex67p (TAP in metazoans), which mediates the interaction between the mRNP and components of the nuclear pore complex (42, 51). The recruitment of Mex67p/TAP to the mRNP is facilitated by Yra1p (Aly in metazoans), an essential mRNA export factor which binds RNA and directly interacts with Mex67p (TAP) (47,48,52 Evidence has been accumulating that the different steps of gene expression, from transcription in the nucleus to translation and degradation in the cytoplasm, are intimately linked (37). Many mRNA processing factors are recruited to growing mRNPs through an interaction with the transcription machinery. For example, the yeast mRNA capping enzymes required for the addition of the m 7 G cap structure associate with the C-terminal domain (CTD) of RNA polymerase II at an early stage of transcription (23). Similarly, components of the splicing or polyadenylation machinery associate with the CTD, thereby positioning them to mediate efficient RNA processing.These interactions are required in vivo for maximum levels of splicing and polyadenylation, as well as for efficient transcription termination in vivo (2,13,17,38).In yeast, defects in 3Ј-end processing and/or polyadenylation prevent mRNA export and lead to the retention of transcripts at or close to their site of transcription, indicating a coupling between 3Ј-end formation and mRNA export in a step that involves the release of mRNA from n...