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The association between hypercholesterolemia and elevated serum apolipoprotein B (APOB) has generated interest in APOB as a therapeutic target for patients at risk of developing cardiovascular disease. In the clinic, mipomersen, an antisense oligonucleotide (ASO) APOB inhibitor, was associated with a trend toward increased hepatic triglycerides, and liver steatosis remains a concern. We found that siRNA-mediated knockdown of ApoB led to elevated hepatic triglycerides and liver steatosis in mice engineered to exhibit a human-like lipid profile. Many genes required for fatty acid synthesis were reduced, suggesting that the observed elevation in hepatic triglycerides is maintained by the cell through fatty acid uptake as opposed to fatty acid synthesis. Fatty acid transport protein 5 (Fatp5/Slc27a5) is required for long chain fatty acid (LCFA) uptake and bile acid reconjugation by the liver. Fatp5 knockout mice exhibited lower levels of hepatic triglycerides due to decreased fatty acid uptake, and shRNA-mediated knockdown of Fatp5 protected mice from diet-induced liver steatosis. Here, we evaluated if siRNA-mediated knockdown of Fatp5 was sufficient to alleviate ApoB knockdown-induced steatosis. We determined that, although Fatp5 siRNA treatment was sufficient to increase the proportion of unconjugated bile acids 100-fold, consistent with FATP5's role in bile acid reconjugation, Fatp5 knockdown failed to influence the degree, zonal distribution, or composition of the hepatic triglycerides that accumulated following ApoB siRNA treatment. Electronic supplementary material The online version of this article (doi:10.1007/s11745-011-3596-3) contains supplementary material, which is available to authorized users.

Chemically stabilized small interfering RNA (siRNA) can be delivered systemically by intravenous injection of lipid nanoparticles (LNPs) in rodents and primates. The biodistribution and kinetics of LNP–siRNA delivery in mice at organ and cellular resolution have been studied using immunofluorescence (IF) staining and quantitative polymerase chain reaction (qPCR). At 0.5 and 2 hr post tail vein injection of Cy5-labeled siRNA encapsulated in LNP, the organ rank-order of siRNA levels is liver > spleen > kidney, with only negligible accumulation in duodenum, lung, heart, and brain. Similar conclusions were drawn by using qPCR to measure tissue siRNA levels as a secondary end point. siRNA levels in these tissues decreased by more than 10-fold after 24 hr. Within the liver, LNPs delivered siRNA to hepatocytes, Kupffer cells, and sinusoids in a time-dependent manner, as revealed by IF staining and signal quantitation methods established using OPERA/Columbus software. siRNA first accumulated in liver sinusoids and trafficked to hepatocytes by 2 hr post dose, corresponding to the onset of target mRNA silencing. Fluorescence in situ hybridization methods were used to detect both strands of siRNA in fixed tissues. Collectively, the authors have implemented a platform to evaluate biodistribution of siRNA across cell types and across tissues in vivo, with the objective of elucidating the pharmacokinetic and pharmacodynamic relationship to guide optimization of delivery vehicles.

Reducing circulating LDL-cholesterol (LDL-c) reduces the risk of cardiovascular disease in people with hypercholesterolemia. Current approaches to reduce circulating LDL-c include statins, which inhibit cholesterol synthesis, and ezetimibe, which blocks cholesterol absorption. Both elevate serum PCSK9 protein levels in patients, which could attenuate their efficacy by reducing the amount of cholesterol cleared from circulation. To determine whether PCSK9 inhibition could enhance LDL-c lowering of both statins and ezetimibe, we utilized small interfer­ing RNAs (siRNAs) to knock down Pcsk9, together with ezetimibe, rosuvastatin, and an ezetimibe/rosuvastatin combination in a mouse model with a human-like lipid profile. We found that ezetimibe, rosuvastatin, and ezetimibe/rosuvastatin combined lower serum cholesterol but induce the expression of Pcsk9 as well as the Srebp-2 hepatic cholesterol biosynthesis pathway. Pcsk9 knockdown in combination with either treatment led to greater reductions in serum non-HDL with a near-uniform reduction of all LDL-c subfractions. In addition to reducing serum cholesterol, the combined rosuvastatin/ezetimibe/Pcsk9 siRNA treatment exhibited a significant reduction in serum APOB protein and triglyceride levels. Taken together, these data provide evidence that PCSK9 inhibitors, in combination with current therapies, have the potential to achieve greater reductions in both serum cholesterol and triglycerides.

Deeper knowledge of pharmacokinetic and pharmacodynamic (PK/PD) concepts for RNA therapeutics is important to streamline the drug development process and for rigorous selection of best performing drug candidates. Here we characterized the PK/PD relationship for small interfering RNAs (siRNAs) targeting luciferase by examining siRNA concentration in plasma and liver, the temporal RNA-induced silencing complex binding profiles, mRNA reduction, and protein inhibition measured by noninvasive bioluminescent imaging. A dose-dependent and time-related decrease in bioluminescence was detected over 25 days after a single treatment of a lipid nanoparticle-formulated siRNA targeting luciferase messenger RNA. A direct relationship was observed between the degree of in vivo mRNA and protein reduction and the Argonaute2 (Ago2)-bound siRNA fraction but not with the total amount of siRNA found in the liver, suggesting that the Ago2-siRNA complex is the key determinant of target inhibition. These observations were confirmed for an additional siRNA that targets endogenously expressed Sjögren syndrome antigen B (Ssb) mRNA, indicating that our observations are not limited to a transgenic mouse system. Our data provide detailed information of the temporal regulation of siRNA liver delivery, Ago2 loading, mRNA reduction, and protein inhibition that are essential for the rapid and cost-effective clinical development of siRNAs therapeutics.

Effective small interfering RNA (siRNA)–mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem–loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.

The use of short interfering RNAs (siRNA) in animals for target validation or as potential therapeutics is hindered by the short physical half-life when delivered as unencapsulated material and in turn the short active half-life of siRNAs in vivo. Here we demonstrate that the character of the two 3'-overhang nucleotides of the guide strand of siRNAs is a determinant of the duration of silencing by siRNAs both in vivo and in tissue culture cells. We demonstrate that deoxyribonucleotides in the guide strand overhang of siRNAs have a negative impact on maintenance of both the in vitro and in vivo activity of siRNAs over time. Overhangs that contain ribonucleotides or 2'-O-methyl modified nucleotides do not demonstrate this same impairment. We also demonstrate that the sequence of an siRNA is a determinant of the duration of silencing of siRNAs directed against the same target even when those siRNAs have equivalent activities in vitro. Our experiments have determined that a measurable duration parameter exists, distinct from both maximum silencing ability and the potency of siRNAs. Our findings provide information on incorporating chemically modified nucleotides into siRNAs for potent, durable therapeutics and also inform on methods used to select siRNAs for therapeutic and research purposes.

The rapid identification of protein biomarkers in biofluids is important to drug discovery and development. Here, we describe a general proteomic approach for the discovery and identification of proteins that exhibit a statistically significant difference in abundance in cerebrospinal fluid (CSF) before and after pharmacological intervention. This approach, differential mass spectrometry (dMS), is based on the analysis of full scan mass spectrometry data. The dMS workflow does not require complex mixing and pooling strategies, or isotope labeling techniques. Accordingly, clinical samples can be analyzed individually, allowing the use of longitudinal designs and within-subject data analysis in which each subject acts as its own control. As a proof of concept, we performed multifactorial dMS analyses on CSF samples drawn at 6 time points from n = 6 cisterna magna ported (CMP) rhesus monkeys treated with 2 potent gamma secretase inhibitors (GSI) or comparable vehicle in a 3-way crossover study that included a total of 108 individual CSF samples. Using analysis of variance and statistical filtering on the aligned and normalized LC-MS data sets, we detected 26 features that were significantly altered in CSF by drug treatment. Of those 26 features, which belong to 10 distinct isotopic distributions, 20 were identified by MS/MS as 7 peptides from CD99, a cell surface protein. Six features from the remaining 3 isotopic distributions were not identified. A subsequent analysis showed that the relative abundance of these 26 features showed the same temporal profile as the ELISA measured levels of CSF A beta 42 peptide, a known pharmacodynamic marker for gamma-secretase inhibition. These data demonstrate that dMS is a promising approach for the discovery, quantification, and identification of candidate target engagement biomarkers in CSF.

Despite recent progress, systemic delivery remains the major hurdle for development of safe and effective small inhibitory RNA (siRNA)–based therapeutics. Encapsulation of siRNA into liposomes is a promising option to overcome obstacles such as low stability in serum and inefficient internalization by target cells. However, a major liability of liposomes is the potential to induce an acute inflammatory response, thereby increasing the risk of numerous adverse effects. In this study, we characterized a liposomal siRNA delivery vehicle, LNP201, which is capable of silencing an mRNA target in mouse liver by over 80%. The biodistribution profile, efficacy after single and multiple doses, mechanism of action, and inflammatory toxicity are characterized for LNP201. Furthermore, we demonstrate that the glucocorticoid receptor (GR) agonist dexamethasone (Dex) inhibits LNP201-induced cytokine release, inflammatory gene induction, and mitogen-activated protein kinase (MAPK) phosphorylation in multiple tissues. These data present a possible clinical strategy for increasing the safety profile of siRNA-based drugs while maintaining the potency of gene silencing.

Disease modifying treatments for Alzheimer's disease (AD) constitute a major goal in medicine. Current trends suggest that biomarkers reflective of AD neuropathology and modifiable by treatment would provide supportive evidence for disease modification. Nevertheless, a lack of quantitative tools to assess disease modifying treatment effects remains a major hurdle. Cerebrospinal fluid (CSF) biochemical markers such as total tau, p-tau and Ab42 are well established markers of AD; however, global quantitative biochemical changes in CSF in AD disease progression remain largely uncharacterized. Here we applied a high resolution open discovery platform, dMS, to profile a cross-sectional cohort of lumbar CSF from post-mortem diagnosed AD patients versus those from non-AD/non-demented (control) patients. Multiple markers were identified to be statistically significant in the cohort tested. We selected two markers SME-1 (p

The use of inbred strains of mice to dissect the genetic complexity of common diseases offers a viable alternative to human studies, given the control over experimental parameters that can be exercised. Central to efforts to map susceptibility loci for common diseases in mice is a comprehensive map of DNA variation among the common inbred strains of mice. Here we present one of the most comprehensive high-density, single nucleotide polymorphism (SNP) maps of mice constructed to date. This map consists of 10,350 SNPs genotyped in 62 strains of inbred mice . We demonstrate the utility of these data via a novel integrative genomics approach to mapping susceptibility loci for complex traits. By integrating in silico quantitative trait locus (QTL) mapping with progressive QTL mapping strategies in segregating mouse populations that leverage large-scale mapping of the genetic determinants of gene expression traits, we not only facilitate identification of candidate quantitative trait genes, but also protect against spurious associations that can arise in genetic association studies due to allelic association among unlinked markers. Application of this approach to our high-density SNP map and two previously described F2 crosses between strains C57BL/6J (B6) and DBA/2J and between B6 ApoE −/− and C3H/HeJ ApoE−/− results in the identification of Insig2 as a strong candidate susceptibility gene for total plasma cholesterol levels.

The reconstruction of genetic networks in mammalian systems is one of the primary goals in biological research, especially as such reconstructions relate to elucidating not only common, polygenic human diseases, but living systems more generally. Here we propose a novel gene network reconstruction algorithm, derived from classic Bayesian network methods, that utilizes naturally occurring genetic variations as a source of perturbations to elucidate the network. This algorithm incorporates relative transcript abundance and genotypic data from segregating populations by employing a generalized scoring function of maximum likelihood commonly used in Bayesian network reconstruction problems. The utility of this novel algorithm is demonstrated via application to liver gene expression data from a segregating mouse population. We demonstrate that the network derived from these data using our novel network reconstruction algorithm is able to capture causal associations between genes that result in increased predictive power, compared to more classically reconstructed networks derived from the same data.

The eukaryotic initiation factor 4G (eIF4G) is the core of a multicomponent switch controlling gene expression at the level of translation initiation. It interacts with the small ribosomal subunit interacting protein, eIF3, and the eIF4E/cap-mRNA complex in order to load the ribosome onto mRNA during cap-dependent translation. We describe the solution structure of the complex between yeast eIF4E/cap and eIF4G (393–490). Binding triggers a coupled folding transition of eIF4G (393–490) and the eIF4E N terminus resulting in a molecular bracelet whereby eIF4G (393–490) forms a right-handed helical ring that wraps around the N terminus of eIF4E. Cofolding allosterically enhances association of eIF4E with the cap and is required for maintenance of optimal growth and polysome distributions in vivo. Our data explain how mRNA, eIF4E, and eIF4G exists as a stable mRNP that may facilitate multiple rounds of ribosomal loading during translation initiation, a key determinant in the overall rate of protein synthesis.

We describe a one-step affinity method for purifying ribosomes from the budding yeast Saccharomyces cerevisiae. Extracts from yeast strains expressing only C-terminally tagged Rpl25 protein or overexpressing this protein in the presence of endogenous Rpl25p were used as the starling materials. The purification was specific for tagged 60S subunits, and resulted in the copurification of 80S subunits and polysomes, as well as ribosome-associated proteins and mRNAs. Two of these associated proteins, Mpt4p and Asc1p, were nearly stoichiometrically bound to the ribosome. In addition, the degree of mRNA association with the purified ribosomes was found to reflect the mRNA's translational status within the cell. The one-step purification of ribosome and its associated components from a crude extract should provide an important tool for future structural and biochemical studies of the ribosome, as well as for expression profiling of translated mRNAs.

The eukaryotic cap and poly(A) tail binding proteins, eIF4E and Pab1p, play important roles in the initiation of protein synthesis. The recent structures of the complex of eIF4E bound to the methylated guanosine (cap) found at the 5'end of messenger RNA (mRNA), the complex of eIF4E bound to peptide fragments of two related translation factors (eIF4G and 4E-BP1), and the complex of the N-terminal fragment of Pab1p bound to polyadenylate RNA have revealed that eIF4E and Pab1p contain at least two distinct functional surfaces. One surface is used for binding mRNA, and the other for binding proteins involved in translation initiation.

The recruitment of the 40S ribosomal subunit to the initiator codon of a eukaryotic mRNA is mediated by the interplay of several different translation initiation complexes (reviewed in reference 18). A key complex in this process is eukaryotic translation initiation factor 4F (eIF4F). The eIF4F complex in higher eukaryotes is composed of three proteins, eIF4G, eIF4A, and eIF4E (7). The eIF4E protein binds to the m7G cap structure at the 5′ end of the mRNA, as well as to eIF4G. The eIF4G protein acts as a bridging factor in that it binds to eIF4E, to the poly(A) tail binding protein Pab1p, and to eIF3, a complex of proteins which is tightly associated with the 40S ribosomal subunit (11, 13, 19, 20, 23). The RNA-stimulated ATPase eIF4A is also associated with eIF4G in higher eukaryotes and is thought to use its ATPase activity to unwind RNA secondary structure in the 5′ leader of each message (21). This presumably allows for the unimpeded scanning of the 40S ribosomal subunit along the 5′ leader as it searches for the initiator codon. The mechanism by which the eIF4F complex mediates translation initiation in the yeast Saccharomyces cerevisiae is slightly more obscure. This organism encodes the eIF4E protein with the CDC33 gene (3), the eIF4A protein with two different genes (TIF1 and TIF2) (17), and the two eIF4G proteins (eIF4G1 and eIF4G2) with the TIF4631 and TIF4632 genes, respectively (8). Although S. cerevisiae expresses all of these putative components of eIF4F, the yeast eIF4A protein has not previously been shown to bind to eIF4G. For example, the purification of eIF4E from higher eukaryotes by cap analog chromatography results in the copurification of eIF4A and eIF4G (reviewed in reference 7), whereas purification of yeast eIF4E through a similar procedure results in the copurification of eIF4G but not eIF4A (9, 26). Furthermore, the initial mapping of the eIF4A binding domain in rabbit eIF4G placed it within the C-terminal third of the protein (16). The yeast eIF4G protein, although homologous to much of mammalian eIF4G, lacks homology to this entire C-terminal region (8). Each of these observations has brought into question whether eIF4A and eIF4G associate in yeast and, more generally, whether the mechanism by which translation initiation occurs in yeast involves the early recruitment of eIF4A to the 5′ end of the mRNA. During the course of our study it was reported that mammalian eIF4G contains a second eIF4A binding site that is distinct from its previously identified C-terminal binding site (14). This site lies in the middle of the protein, which is conserved in all eukaryotic eIF4G proteins. It was shown in that study that the presence of both eIF4A binding sites on mammalian eIF4G was required for eIF4G to stimulate translation in vitro. Based on extensive sequence homology between yeast and mammalian eIF4G proteins, it was also proposed that yeast eIF4A binds to eIF4G within this region. Here we present evidence that yeast eIF4A and eIF4G functionally and physically interact. Overexpression of eIF4A in several different tif4632 mutants led to suppression of their temperature-sensitive phenotype. This suppression was shown to be allele specific. A heat-inactivated translation extract from a tif4632 mutant could have its translational activity significantly stimulated by the addition of excess eIF4A, and this stimulation was also shown to be tif4632 allele specific. Finally, the binding of recombinant yeast eIF4A to eIF4G1 and eIF4G2 in vitro was demonstrated for the first time, as was a decreased ability of the several mutant eIF4G2 proteins to bind to eIF4A. These data show that yeast eIF4G contains an eIF4A binding site, and they indicate that an interaction between yeast eIF4A and eIF4G is required for translation and is essential for yeast cell viability.

Interaction of the poly(A) binding protein, Pab1p, with mRNA plays an important role in gene expression. This work describes an analysis of pab1 mutants in Saccharomyces cerevisiae. Yeast pab1 mutants were found to be sensitive to elevated concentrations of copper (Cu) and 3-aminotriazole (3-AT) in the growth medium. This phenotype arises because these pab1 mutants underaccumulate mRNA, including the CUP1 and HIS3 mRNAs, the products of which are required for Cu and 3-AT resistance, respectively. To determine the cause of the mRNA underaccumulation, mRNA turnover and production were examined in the pab1-53 mutant. It was found that although the pattern of mRNA decay was altered, and substantial decapping of polyadenylated mRNA could be detected, mRNA was not destabilized in this strain. It was also found that the pab1 mutant was impaired in the production of mRNA. These data show that the decreased level of mRNA in the pab1-53 mutant arises from poor production, and they suggest that yeast Pab1p is involved in an aspect of nuclear mRNA metabolism. They also indicate that deadenylation can be uncoupled from decapping without significant changes in an mRNA's stability, and that this uncoupling can be tolerated by yeast. Copyright © 1999 John Wiley & Sons, Ltd.

Translation initiation in extracts from Saccharomyces cerevisiae involves the concerted action of the cap-binding protein eIF4E and the poly(A) tail-binding protein Pab1p. These two proteins bind to translation initiation factor eIF4G and are needed for the translation of capped or polyadenylated mRNA, respectively. Together, these proteins synergistically activate the translation of a capped and polyadenylated mRNA. We have discovered that excess Pab1p also stimulates the translation of capped mRNA in extracts, a phenomenon that we define as trans-activation. Each of the above activities of Pab1p requires its second RNA recognition motif (RRM2). We have found that RRM2 from human PABP cannot substitute functionally for yeast RRM2. Using the differences between human and yeast RRM2 sequences as a guide, we have mutagenized yeast RRM2 and discovered residues that are required for eIF4G binding and poly(A)-dependent translation but not for trans-activation. Similarly, other residues within RRM2 were found to be required for trans-activation but not for eIF4G binding or poly(A)-dependent translation. These data show that Pab1p has at least two biochemically distinct activities in translation extracts.

We report that newly synthesized mRNA poly(A) tails are matured to precise lengths by the Pab1p-dependent poly(A) nuclease (PAN) of Saccharomyces cerevisiae. These results provide evidence for an initial phase of mRNA deadenylation that is required for poly(A) tail length control. In RNA 3′-end processing extracts lacking PAN, transcripts are polyadenylated to lengths exceeding 200 nucleotides. By contrast, in extracts containing PAN, transcripts were produced with the expected wild-type poly(A) tail lengths of 60 to 80 nucleotides. The role for PAN in poly(A) tail length control in vivo was confirmed by the finding that mRNAs are produced with longer poly(A) tails in PAN-deficient yeast strains. Interestingly, wild-type yeast strains were found to produce transcripts which varied in their maximal poly(A) tail length, and this message-specific length control was lost in PAN-deficient strains. Our data support a model whereby mRNAs are polyadenylated by the 3′-end processing machinery with a long tail, possibly of default length, and then in a PAN-dependent manner, the poly(A) tails are rapidly matured to a message-specific length. The ability to control the length of the poly(A) tail for newly expressed mRNAs has the potential to be an important posttranscriptional regulatory step in gene expression.

Communication between the 5' cap structure and 3' poly(A) tail of eukaryotic mRNA results in the synergistic enhancement of translation. The cap and poly(A) tail binding proteins, eIF4E and Pab1p, mediate this effect in the yeast S. cerevisiae through their interactions with different parts of the translation factor eIF4G. Here, we demonstrate the reconstitution of an eIF4E/eIF4G/Pab1p complex with recombinant proteins, and show by atomic force microscopy that the complex can circularize capped, polyadenylated RNA. Our results suggest that formation of circular mRNA by translation factors could contribute to the control of mRNA expression in the eukaryotic cell.

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.

The exclusive role of the mRNA cap structure in the 40S subunit recruitment process during translation initiation is no longer tenable. The ability of IRES elements and poly(A) tails in yeast to stimulate 40S subunit binding forces a change in this viewpoint. While it is almost certain that the vast majority of mRNAs are translated by 40S subunits scanning from the cap structure, it now seems likely that the 40S subunit can also be brought to the mRNA via an interaction with the mRNA poly(A) tail. Subsequently or simultaneously with this interaction, the cap structure, due to its high affinity for eIF4E, could act as a docking site for the recruited subunit (Figure 3Figure 3). In this model, both the cap structure and the poly(A) tail share the function of 40S subunit recruitment, while the cap structure has the exclusive role of docking the subunit onto a unique position in the mRNA. We note that the role of poly(A) tails in translation in higher eukaryotes is assumed but not yet shown since only the role of poly(A) tails in yeast translation has been thoroughly studied.Cellular IRES elements could replace the role of the cap structure on those mRNAs where the cap is either masked or eIF4E is inactive (Figure 3Figure 3). Recruitment of the 40S subunit to the mRNA by the poly(A) tail could occur prior to or simultaneously with the placement of the subunit at a position on the mRNA determined by the location of the IRES element. In this model, the cap structure and the IRES element have identical functions in the translation process: they assist in recruitment of the 40S subunit to the mRNA, and they provide a loading site for 40S subunits at a unique position on the mRNA.Future work in this exciting area of translation research should help to test the basic tenets of this model. Since many of the central experiments so far have been carried out in cell-free systems and commonly under conditions where mRNA is limiting, it will be important to determine the interplay of the diferent modes of ribosome recruitment under conditions of mRNA competition for initiation factors. Likewise, we do not yet understand much regarding possible differences between the first and subsequent rounds of initiation, and the roles of the cap, IRES and poly(A) tail in ribosome recycling. Along the way, it is anticipated that new insights into how an mRNA's expression can be controlled in the cytoplasm of eukaryotic cells will be uncovered. With more information, it will hopefully become clearer how mRNA sequences, including the 5′ NCR and the 3′ UTR, can regulate an mRNA's expression.

The yeast poly(A)-binding protein (Pab1p) contains four RNA recognition motifs (RRMs). Site-directed mutations were introduced into each of these RRMs in order to investigate their relative contributions to specific and non-specific RNA binding, and to determine the consequences of these mutations on the ability of Pab1p to support viability. Specifically, a charged and an aromatic residue that were predicted to be involved in RNA binding were mutated in each RRM. These mutations revealed that the second RRM is primarily responsible for poly(A) binding, while the fourth RRM is primarily responsible for non-specific polypyrimidine RNA binding. The mutated aromatic residues in each RRM contributed to both modes of binding whereas the mutated charged residues contributed primarily to non-specific RNA binding. RNA binding in vivo correlated with the in vitro binding measurements. Furthermore, RNA binding, but not high-affinity poly(A) binding, correlated with the ability of Pab1p to sustain yeast cell viability. These data suggest that a single aromatic substitution in Pab1p can significantly reduce its RNA binding ability, that the capacity of Pab1p to bind poly(A) as well as other RNAs is mediated by distinct residues within different RRMs, and that Pab1p does not require high affinity poly(A) tail binding to perform its essential function.

A major hurdle for harnessing small interfering RNA (siRNA) for therapeutic application is an effective and safe delivery of siRNA to target tissues and cells via systemic administration. While lipid nanoparticles (LNPs) composed of a cationic lipid, poly-(ethylene glycol) lipid and cholesterol, are effective in delivering siRNA to hepatocytes via systemic administration, they may induce multi-faceted toxicities in a dose-dependent manner, independently of target silencing. To understand the underlying mechanism of toxicities, pharmacological probes including anti-inflammation drugs and specific inhibitors blocking different pathways of innate immunity were evaluated for their abilities to mitigate LNP-siRNA-induced toxicities in rodents. Three categories of rescue effects were observed: (i) pretreatment with a Janus kinase (Jak) inhibitor or dexamethasone abrogated LNP-siRNA-mediated lethality and toxicities including cytokine induction, organ impairments, thrombocytopenia and coagulopathy without affecting siRNA-mediated gene silencing; (ii) inhibitors of PI3K, mammalian target of rapamycin (mTOR), p38 and IκB kinase (IKK)1/2 exhibited a partial alleviative effect; (iii) FK506 and etoricoxib displayed no protection. Furthermore, knockout of Jak3, tumor necrosis factor receptors (Tnfr)p55/p75, interleukin 6 (IL-6) or interferon (IFN)-γ alone was insufficient to alleviate LNP-siRNA-associated toxicities in mice. These indicate that activation of innate immune response is a primary trigger of systemic toxicities and that multiple innate immune pathways and cytokines can mediate toxic responses. Jak inhibitors are effective in mitigating LNP-siRNA-induced toxicities.

Poly(A) tail removal is the first step in the degradation pathway for some mRNAs. The purified poly(A)-binding protein (PAB)-dependent poly(A) nuclease (PAN) from yeast removes mRNA poly(A) tails in vitro by a process similar to that observed in vivo. The exonucleolytic PAN degrades poly(A) and RNA bound by PAB, and can be activated by spermidine to degrade poly(A) in the absence of PAB. The shortening of the poly(A) tail down to 10-25 nucleotides and the terminal deadenylation of this short adenine tract are kinetically distinct reactions. Poly(A) shortening rates are stimulated by the yeast a-mating factor (MFA2) RNA 3' UTR sequence, and this occurs by switching PAN from a distributive to a more processive enzyme. Terminal deadenylation rates are also stimulated to different extents by various RNAs. Inversion of the MFA2 3' UTR sequence completely inhibits the terminal deadenylation reaction owing to the presence of an inhibitory element 70 nucleotides from the poly(A) tail. Other sequence elements inserted at a similar distance from the poly(A) tail also interfere with the reaction. These data suggest that the two phases of poly(A) degradation can be regulated by mRNA sequences, and they provide a mechanistic description of how this regulation could occur in vivo.

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2', 3' seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5'-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5'-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.

Corrigendum to “Integrating QTL and high-density SNP analyses in mice to identify Insig2 as a susceptibility gene for plasma cholesterol levels” [Genomics 86 (2005) 505–517]☆ Alessandra C. Cervino , Guoya Li , Steve Edwards , Jun Zhu , Cathy Laurie , George Tokiwa , Pek Yee Lum , Susanna Wang , Lawrence W. Castellani , Aldons J. Lusis , Sonia Carlson , Alan B. Sachs , Eric E. Schadt a,⁎ a Rosetta Inpharmatics, LLC, a Wholly Owned Subsidiary of Merck and Co., Inc., Seattle, WA 98109, USA b Division of Cardiology, Department of Medicine, and Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at the University of California at Los Angeles, Los Angeles, CA 90095-1679, USA

siRNAs mediate sequence-specific gene silencing in cultured mammalian cells but also silence unintended transcripts. Many siRNA off-target transcripts match the guide-strand “seed region,” similar to the way microRNAs match their target sites. The extent to which this seed-matched, microRNA-like, off-target silencing affects the specificity of therapeutic siRNAs in vivo is currently unknown. Here, we compare microRNA-like off-target regulations in mouse liver in vivo with those seen in cell culture for a series of therapeutic candidate siRNAs targeting Apolipoprotein B (APOB). Each siRNA triggered regulation of consistent microRNA-like off-target transcripts in mouse livers and in cultured mouse liver tumor cells. In contrast, there was only random overlap between microRNA-like off-target transcripts from cultured human and mouse liver tumor cells. Therefore, siRNA therapeutics may trigger microRNA-like silencing of many unintended targets in vivo, and the potential toxicities caused by these off-target gene regulations cannot be accurately assessed in rodent models.

Rare familial forms of Alzheimer's disease (AD) are thought to be caused by elevated proteolytic production of the Aβ42 peptide from the β-amyloid-precursor protein (APP). Although the pathogenesis of the more common late-onset AD (LOAD) is not understood, BACE1, the protease that cleaves APP to generate the N terminus of Aβ42, is more active in patients with LOAD, suggesting that increased amyloid production processing might also contribute to the sporadic disease. Using high-throughput siRNA screening technology, we assessed 15,200 genes for their role in Aβ42 secretion and identified leucine-rich repeat transmembrane 3 ( LRRTM3 ) as a neuronal gene that promotes APP processing by BACE1. siRNAs targeting LRRTM3 inhibit the secretion of Aβ40, Aβ42, and sAPPβ, the N-terminal APP fragment produced by BACE1 cleavage, from cultured cells and primary neurons by up to 60%, whereas overexpression increases Aβ secretion. LRRTM3 is expressed nearly exclusively in the nervous system, including regions affected during AD, such as the dentate gyrus. Furthermore, LRRTM3 maps to a region of chromosome 10 linked to both LOAD and elevated plasma Aβ42, and is structurally similar to a family of neuronal receptors that includes the NOGO receptor, an inhibitor of neuronal regeneration and APP processing. Thus, LRRTM3 is a functional and positional candidate gene for AD, and, given its receptor-like structure and restricted expression, a potential therapeutic target.

The identification of genes and, more generally, gene networks underlying common human diseases remain one of the primary objectives in the biomedical and life sciences. Success in reconstructing gene networks underlying complex physiological traits and identifying the key drivers of these traits will largely rest on integrative approaches that combine data from multiple different sources. Key to this integration will be the development and application of sophisticated algorithms to mine the diversity of data. Here we review an integrative genomics approach that enhances the utility of genetics approaches commonly used to dissect complex traits. This integrative genomics approach takes into account genotypic, molecular profiling, and clinical data in segregating populations to identify the key drivers of disease and reconstruct disease-specific gene networks. Keywords: genetics of gene expression; QTL; eQTL; gene networks; common disease; complex traits; integrative genomics

Niemann-Pick C1-like 1 (NPC1L1) is an intestinal cholesterol transporter and the molecular target of ezetimibe, a cholesterol absorption inhibitor demonstrated to reduce LDL-cholesterol (LDL-C) both as monotherapy and when co-administered with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins). Interestingly, significant interindividual variability has been observed for rates of intestinal cholesterol absorption and LDL-C reductions at both baseline and post ezetimibe treatment. To test the hypothesis that genetic variation in NPC1L1 could influence the LDL-C response to ezetimibe, we performed extensive resequencing of the gene in 375 apparently healthy individuals and genotyped hypercholesterolemic patients from clinical trial cohorts. No association was observed between NPC1L1 single-nucleotide polymorphism and baseline cholesterol. However, significant associations to LDL-C response to treatment with ezetimibe were observed in patients treated with ezetimibe in two large clinical trials. Our data demonstrate that DNA sequence variants in NPC1L1 are associated with an improvement in response to ezetimibe pharmacotherapy and suggest that detailed analysis of genetic variability in clinical trial cohorts can lead to improved understanding of factors contributing to variable drug response.

The integration of biomarkers reporting on drug pharmacodynamics or on a disease state is becoming necessary in the cost-effective design of clinical trials. Transcriptional changes in skin measured using whole-genome arrays have been useful in assessing the disease state in dermatology. Not only are skin biopsies well-tolerated and easy to obtain, but they sample a system in which many complex signaling and developmental networks are active and in which a wide variety of drug targets may be expected to play a role, either directly or indirectly. Recent advances have led to the use of transcriptional profiling of skin for making pharmacodynamic measurements in clinical trials, even for non-dermatological conditions.

Drugs designed against targets in presumably simple linear signaling pathways found to be associated with disease are often less effective than predicted. One reason for this is the overly simplistic view of the molecular mechanisms underlying common human diseases. This viewpoint is a consequence of biological reductionism, brought about by the need to form a basic understanding of the fundamental attributes of biological systems and by limitations in the set of tools available for analysis of biological systems. However, complex biological systems are best modeled as highly modular, fluid systems exhibiting a plasticity that allows them to adapt to a vast array of conditions. Historically, this viewpoint has long represented the ideal, but the tools needed to examine and describe this complexity were often lacking. Here we argue that the tools of biological science now allow for a more network-oriented view of biological systems and for explaining the underlying causes of disease, as well as the best ways to target disease. Ultimately, this will help to ensure that the right drug is administered to the right patient at the right time. Focusing on well-studied signaling pathways, refining the definition of disease, and identifying disease subtypes, we demonstrate a more holistic approach to elucidating common human diseases, with the potential to revolutionize treatment of these diseases.

A key goal of biomedical research is to elucidate the complex network of gene interactions underlying complex traits such as common human diseases. Here we detail a multistep procedure for identifying potential key drivers of complex traits that integrates DNA-variation and gene-expression data with other complex trait data in segregating mouse populations. Ordering gene expression traits relative to one another and relative to other complex traits is achieved by systematically testing whether variations in DNA that lead to variations in relative transcript abundances statistically support an independent, causative or reactive function relative to the complex traits under consideration. We show that this approach can predict transcriptional responses to single gene–perturbation experiments using gene-expression data in the context of a segregating mouse population. We also demonstrate the utility of this approach by identifying and experimentally validating the involvement of three new genes in susceptibility to obesity.

The cytoplasmic fate of mRNAs is dictated by the relative activities of the intimately connected mRNA decay and translation initiation pathways. In this study, we have found that yeast strains compromised for stages downstream of deadenylation in the major mRNA decay pathway are incapable of inhibiting global translation initiation in response to stress. In the past, the paradigm of the eIF2alpha kinase-dependent amino acid starvation pathway in yeast has been used to evaluate this highly conserved stress response in all eukaryotic cells. Using a similar approach we have found that even though the mRNA decay mutants maintain high levels of general translation, they exhibit many of the hallmarks of amino acid starvation, including increased eIF2alpha phosphorylation and activated GCN4 mRNA translation. Therefore, these mutants appear translationally oblivious to decreased ternary complex abundance, and we propose that this is due to higher rates of mRNA recruitment to the 40S ribosomal subunit.

Fusel alcohols are natural products of amino acid catabolism in the yeast Saccharomyces cerevisiae that cause morphological changes similar to those seen during pseudohyphal growth. We have discovered that certain of these alcohols, including butanol and isoamyl alcohol, bring about a rapid inhibition of translation at the initiation step. This inhibition is strain specific and is not explained by previously described translational control pathways. Using genetic mapping, we have identified a proline to serine allelic variation at amino acid 180 of the GCD1 gene product as the genetic locus that allows translational regulation upon butanol addition. Gcd1p forms part of the eIF2B guanine nucleotide complex that is responsible for recycling eIF2‐GDP to eIF2‐GTP. This represents one of the key limiting steps of translation initiation and we provide evidence that fusel alcohols target eIF2B in order to bring about translational regulation.

Here we report that the Saccharomyces cerevisiae RBP29 (SGN1, YIR001C) gene encodes a 29-kDa cytoplasmic protein that binds to mRNA in vivo. Rbp29p can be co-immunoprecipitated with the poly(A) tail-binding protein Pab1p from crude yeast extracts in a dosage- and RNA-dependent manner. In addition, recombinant Rbp29p binds preferentially to poly(A) with nanomolar binding affinity in vitro. Although RBP29 is not essential for cell viability, its deletion exacerbates the slow growth phenotype of yeast strains harboring mutations in the eIF4G genes TIF4631 and TIF4632. Furthermore, overexpression of RBP29 suppresses the temperature-sensitive growth phenotype of specific tif4631, tif4632, and pab1 alleles. These data suggest that Rbp29p is an mRNA-binding protein that plays a role in modulating the expression of cytoplasmic mRNA.

Glucose performs key functions as a signaling molecule in the yeastSaccharomyces cerevisiae. Glucose depletion is known to regulate gene expression via pathways that lead to derepression of genes at the transcriptional level. In this study, we have investigated the effect of glucose depletion on protein synthesis. We discovered that glucose withdrawal from the growth medium led to a rapid inhibition of protein synthesis and that this effect was readily reversed upon readdition of glucose. Neither the inhibition nor the reactivation of translation required new transcription. This inhibition also did not require activation of the amino acid starvation pathway or inactivation of the TOR kinase pathway. However, mutants in the glucose repression (reg1, glc7,hxk2, and ssn6), hexose transporter induction (snf3 rgt2), and cAMP-dependent protein kinase (tpk1wandtpk2w) pathways were resistant to the inhibitory effects of glucose withdrawal on translation. These findings highlight the intimate connection between the nutrient status of the cell and its translational capacity. They also help to define a new area of posttranscriptional regulation in yeast.

The function of the poly(A) tail on mRNA in eucaryotes is the subject of much research. It has been shown that in the yeast Saccharomyces cerevisiae, the poly(A) tail acts to enhance the translation of the mRNA (reviewed in reference 36). This activity requires the poly(A)-binding protein Pab1p, which, through an interaction with a protein complex recognizing the cap structure, is thought to stimulate the binding of ribosomes to the 5′ end of the mRNA (23, 39–41). Another potential function of the poly(A) tail is to stabilize mRNA. Several independent observations support this hypothesis. A series of detailed studies of both yeast and mammalian cells has documented that mRNA deadenylation usually precedes mRNA degradation (reviewed in reference 8). More specifically, it has been shown that in yeast, mRNA decapping, the initiating event in the degradation of the majority of mRNAs, occurs after deadenylation (9, 26, 27). Following decapping, these mRNAs are destroyed by the 5′-3′ exoribonuclease Xrn1p (20, 26). It has also been shown that decreasing the rate of poly(A) tail removal by mutagenizing mRNA (for example, see reference 28) results in lower rates of mRNA degradation. Degradation through the pathway of decapping and then digestion by the 5′-3′ exonuclease Xrn1p is not the sole means by which mRNA is degraded in yeast. Three key observations form the basis for this conclusion. First, targeted disruption of either the decapping enzyme gene DCP1 or the exonuclease gene XRN1 does not lead to cell inviability or greater than four- to fivefold stabilization of mRNAs that are normally unstable (4, 20). Second, the disruption of these genes does not change the stability of the most stable yeast mRNAs, such as PGK1 or ACT1, by more than a factor of 2 (4, 27). Finally, for yeast strains containing a disruption of the XRN1 gene or a mutation in the Dcp1 protein, the existence of mRNA species trimmed from the 3′ end has been reported (27). A common feature of both the mRNA translation and degradation reactions is that the cap structure and the poly(A) tail appear to be involved in their regulation (reviewed in reference 42). The possibility that the roles of these two structures in the degradation reaction are functionally linked was supported by the recent report that mutations in the yeast decapping enzyme Dcp1p and the unidentified gene products of the MRT1 and MRT3 genes, which also appear to regulate mRNA decapping rates, can allow yeast cells to survive in the absence of Pab1p (17). These mutations were therefore suggested to allow for cell viability in the absence of Pab1p by stabilizing the mRNA to an extent that allowed for sufficient expression in the absence of Pab1p. We originally chose to further explore the essential roles of Pab1p in yeast in order to define the mechanistic roles of Pab1p and poly(A) in mRNA metabolism (33, 34). A series of genetic suppression experiments identified mutations in other yeast genes which allowed for cell growth in the absence of Pab1p. Most of these mutations resulted in aberrant 60S ribosomal subunit production. Other laboratories have also identified similar types of pab1Δ bypass suppressors (43). To identify proteins involved in mRNA degradation, including those that functionally interact with Pab1p and the poly(A) tail during this process, we chose to focus our newer studies on those pab1Δ bypass suppressor mutations that did not alter the levels of the ribosomal subunits. Based on published data (17), we reasoned that such mutations could lead to viability in the absence of Pab1p by stabilizing the mRNA. The current lack of sequence information on genes other than DCP1 and XRN1 that are known to alter the pathway of 5′-3′ mRNA degradation in yeast provided us with further incentive to pursue this avenue of investigation. Here we report that a null mutation in the nonessential yeast SPB8 (YJL124c) gene leads to bypass suppression of a PAB1 deletion without altering ribosomal subunit levels. Spb8p was found to contain an Sm-like domain. Mutations within Spb8p led to the accumulation of capped, deadenylated degradation intermediates and in some cases the stabilization of mRNA. Cap stabilization was so complete in the spb8-2 strain that 3′-5′ mRNA degradation intermediates also became readily detectable. These data support the hypotheses that Spb8p is needed for normal rates of mRNA decapping in yeast and that Spb8p allows for rates of decapping and 5′-3′ degradation that preclude the detection of 3′-5′ degradative intermediates.

The eukaryotic mRNA 3′ poly(A) tail and its associated poly(A)-binding protein (Pab1p) are important regulators of gene expression. One role for this complex in the yeast Saccharomyces cerevisiae is in translation initiation through an interaction with a 115-amino-acid region of the translation initiation factor eIF4G. The eIF4G-interacting domain of Pab1p was mapped to its second RNA recognition motif (RRM2) in an in vitro binding assay. Moreover, RRM2 of Pab1p was required for poly(A) tail-dependent translation in yeast extracts. An analysis of a site-directed Pab1p mutation which bound to eIF4G but did not stimulate translation of uncapped, polyadenylated mRNA suggested additional Pab1p-dependent events during translation initiation. These results support the model that the association of RRM2 of yeast Pab1p with eIF4G is a prerequisite for the poly(A) tail to stimulate the translation of mRNA in vitro.

The yeast translation factor eIF4G associates with both the cap-binding protein eIF4E and the poly(A)-binding protein Pab1p. Here we report that the two yeast eIF4G homologs, Tif4631p and Tif4632p, share a conserved Pab1p-binding site. This site is required for Pab1p and poly(A) tails to stimulate the in vitro translation of uncapped polyadenylylated mRNA, and the region encompassing it is required for the cap and the poly(A) tail to synergistically stimulate translation. This region on Tif4631p becomes essential for cell growth when the eIF4E binding site on Tif4631p is mutated. Pab1p mutations also show synthetic lethal interactions with eIF4E mutations. These data suggest that eIF4G mediates poly(A) tail stimulated translation in vitro , and that Pab1p and the domain encompassing the Pab1p-binding site on eIF4G can compensate for partial loss of eIF4E function in vivo .

Publisher Summary This chapter discusses about messenger RNA ribonucleases and mRNA turnover in Saccharomyces cerevisiae. Among the many different functions for ribonucleases, one fundamental role is to degrade messenger RNA (mRNA). The regulated turnover of mRNA is critical in the control of gene expression. The stabilization of certain unstable mRNAs encoding protooncogenes can contribute to oncogenesis. In addition, mRNA stability can be regulated in accordance with changes in the cell growth rate, progression through the cell cycle, and changes in the cellular environment. In many cases, mRNA abundance seems to correlate more closely with mRNA half-lives than with rates of transcription. The ability to perform both advanced genetics and biochemistry with S. cerevisiae makes it well suited for dissecting the fundamental mechanisms of mRNA degradation in eukaryotes. In yeast, some mRNAs are degraded with half-lives greater than 40 min, whereas others are degraded 20 times faster. Ultimately, differential mRNA decay is determined by the regulation of cellular mRNA ribonucleases (mRNases). This chapter discusses about the pathways of mRNA decay, deadenylation-dependent decapping, nonsense-mediated decay, and endonuclease-initiated turnover pathway. The chapter also elaborates about mRNA ribonucleases, decapping enzymes, and deadenylases.

The removal of the mRNA poly(A) tail in the yeast Saccharomyces cerevisiae is stimulated by the poly(A)-binding protein (Pab1p). A large scale purification of the Pab1p-stimulated poly(A) ribonuclease (PAN) identifies a 76-kDa and two 135-Da polypeptides as candidate enzyme subunits. Antibodies against the Pan1p protein, which is the minor 135-kDa protein in the preparation, can immunodeplete Pan1p but not PAN activity. The protein sequence of the major 135-kDa protein, Pan2p, reveals a novel protein that was also found in the previously reported PAN purification (Sachs, A. B., and Deardorff, J. A. (1992) Cell 70, 961-973). Deletion of the non-essential PAN2 gene results in an increase of the average length of mRNA poly(A) tails in vivo, and a loss of Pab1p-stimulated PAN activity in crude extracts. These data confirm that Pan2p and not Pan1p is required for PAN activity, and they suggest that ribonucleases other than the Pab1p-stimulated PAN are capable of shortening poly(A) tails in vivo.

The mRNA poly(A) tail and its associated poly(A) binding protein (Pab1p) are ubiquitous in eukaryotes. The function of the poly(A) tail is to stabilize mRNA and to stimulate its translation. The development of a poly(A)- and cap-dependent yeast in vitro translation system has allowed us to understand how poly(A) stimulates translation. We find that Pab1p but not the cap binding protein eIF-4E is required for poly(A) tail-dependent translation, and that the Pab1p-poly(A) tail complex functions to recruit the 40S ribosomal subunit to the mRNA. These data introduce a new step into the pathway of translation initiation and merge the translational functions of the two ends of mRNA.

Messenger RNA translation initiation and cytoplasmic poly(A) tail shortening require the poly(A)-binding protein (PAB) in yeast. The PAB-dependent poly(A) ribonuclease (PAN) has been purified to near homogeneity from S. cerevisiae based upon its PAB requirement, and its gene has been cloned. The essential PAN1 gene encodes a 161 kd protein organized into distinct domains containing repeated sequence elements. Deletion analysis of the gene revealed that only one-third of the protein is needed to maintain cell viability. Conditional mutations in PAN1 lead to an arrest of translation initiation and alterations in mRNA poly(A) tail lengths. These data suggest that PAN could mediate each of the PAB-dependent reactions within the cell, and they provide evidence for a direct relationship between translation initiation and mRNA metabolism.