Eukaryotic Protein-coding Genes Research Articles

Ending the message: poly(A) signals then and now

Polyadenylation [poly(A)] signals (PAS) are a defining feature of eukaryotic protein-coding genes. The central sequence motif AAUAAA was identified in the mid-1970s and subsequently shown to require flanking, auxiliary elements for both 3'-end cleavage and polyadenylation of premessenger RNA (pre-mRNA) as well as to promote downstream transcriptional termination. More recent genomic analysis has established the generality of the PAS for eukaryotic mRNA. Evidence for the mechanism of mRNA 3'-end formation is outlined, as is the way this RNA processing reaction communicates with RNA polymerase II to terminate transcription. The widespread phenomenon of alternative poly(A) site usage and how this interrelates with pre-mRNA splicing is then reviewed. This shows that gene expression can be drastically affected by how the message is ended. A central theme of this review is that while genomic analysis provides generality for the importance of PAS selection, detailed mechanistic understanding still requires the direct analysis of specific genes by genetic and biochemical approaches.

Crystal Structure of a Ten-Subunit Human Spliceosomal U1 snRNP at 5.5 Å Resolution

Most eukaryotic protein-coding genes contain non-coding regions (introns) that separate those coding for protein (exons). The introns must be excised and exons spliced together from the precursor-mRNA transcript of such genes. This process (RNA splicing) is catalyzed by the spliceosome, integral to which are five RNA-protein complexes (U1, U2, U4, U5 and U6 snRNPs). A first step in RNA splicing, functioning to initiate spliceosome assembly, involves recognition of the junction between the 5′-exon and intron (5′-splice site) by U1 snRNP.

Identification and characterization of the activation domain of Ifh1, an activator of model TATA-less genes

In yeast, TATA box-binding protein TBP can be delivered to protein-coding genes by direct interactions with two different coactivators: TFIID, which delivers TBP preferentially to TATA-less promoters, and SAGA, which strongly favors TATA box-containing promoters. Transcriptional activators of SAGA-dependant genes are characterized by prototypic acidic activation domains (ADs) that efficiently recruit SAGA, but not TFIID, to UAS elements even in the absence of a core promoter. In contrast to the well-studied acidic activation domains, little is known about the activation domains of activators of TFIID-dependent genes, even though these genes constitute more than 80% of eukaryotic protein-coding genes. The paradigm for TATA-less genes are the ribosomal protein genes (RPGs). Here we have identified the AD of the RPG activator Ifh1p and demonstrate that a minimal Ifh1 AD represents a new class of AD that significantly differs from acidic ADs in amino acid signature, relative coactivator affinities, and core promoter selectivity.

Integrative Discovery of Multifaceted Sequence Patterns by Frame-Relayed Search and Hybrid PSO-ANN

For de novo pattern mining in genomic sequences, the main issues are constructing pattern definition model (PDM) and mining sequence patterns (MSP). The representations of PDMs and the discovery of patterns are functionally dependent; the performances thus depend on the adopted PDMs. The popular PDMs provide only descriptive patterns; they lack multi- faceted considerations. Many of existing MSP methods are tied up with the exclusively devised PDMs, and the specialized and sophisticated models make the mined results hard to be reused. In this research, an integrative pattern mining system is proposed, which consists of a computation- oriented PDM (CO-PDM) and general-purpose MSP (GP-MSP) methods. The CO-PDM defines four computational concerns (CCs) as facets of MSP: expression (E), location (L), range (R) and weight (W), which are integrated into a frame-relayed pattern model (FRPM). The GP-MSP develops a frame-relayed search strategy to resolve the ELR-CCs firstly, with the aids of critical- parameter automating (CPA) procedure; and then the W-CC is determined by hybridizing par- ticle swarm optimization (PSO) and artificial neural network (ANN). The proposed FRPM and GP-MSP had been implemented and applied to 22,448 human introns; from the results, all the well-known patterns were recovered and some new ones were also discovered. Furthermore, the effectiveness of identified patterns were verified by a two-layered k-nearest neighbor (k-NN) classifier; the average precision and recall are 0.88 and 0.92, respectively. By the case study, the integrative PDM-MSP system is believed to be effective and reliable; it is optimistic the proposed CO-PDM and GP-MSP are both widely applicable and reusable for mining sequence patterns in the eukaryotic protein-coding genes.

The Deep Archaeal Roots of Eukaryotes

The set of conserved eukaryotic protein-coding genes includes distinct subsets one of which appears to be most closely related to and, by inference, derived from archaea, whereas another one appears to be of bacterial, possibly, endosymbiotic origin. The “archaeal” genes of eukaryotes, primarily, encode components of information-processing systems, whereas the “bacterial” genes are predominantly operational. The precise nature of the archaeo–eukaryotic relationship remains uncertain, and it has been variously argued that eukaryotic informational genes evolved from the homologous genes of Euryarchaeota or Crenarchaeota (the major branches of extant archaea) or that the origin of eukaryotes lies outside the known diversity of archaea. We describe a comprehensive set of 355 eukaryotic genes of apparent archaeal origin identified through ortholog detection and phylogenetic analysis. Phylogenetic hypothesis testing using constrained trees, combined with a systematic search for shared derived characters in the form of homologous inserts in conserved proteins, indicate that, for the majority of these genes, the preferred tree topology is one with the eukaryotic branch placed outside the extant diversity of archaea although small subsets of genes show crenarchaeal and euryarchaeal affinities. Thus, the archaeal genes in eukaryotes appear to descend from a distinct, ancient, and otherwise uncharacterized archaeal lineage that acquired some euryarchaeal and crenarchaeal genes via early horizontal gene transfer.

Human Pcf11 enhances degradation of RNA polymerase II-associated nascent RNA and transcriptional termination

The poly(A) (pA) signal possesses a dual function in 3′ end processing of pre-mRNA and in transcriptional termination of RNA polymerase II (Pol II) for most eukaryotic protein-coding genes. A key protein factor in yeast and Drosophila Pol II transcriptional termination is the 3′-end processing factor, Pcf11. In vitro studies suggest that Pcf11 is capable of promoting the dissociation of Pol II elongation complexes from DNA. Moreover, several mutant alleles of yeast Pcf11 effect termination in vivo. However, functions of human Pcf11 (hPcf11) in Pol II termination have not been explored. Here we show that depletion of hPcf11 from HeLa cells reduces termination efficiency. Furthermore, we provide evidence that hPcf11 is required for the efficient degradation of the 3′ product of pA site cleavage. Finally, we show that these functions of hPcf11 require an intact pA signal.

Life beyond cleavage: the case of Ago2 and hematopoiesis

A recent article in The Economist (June 16, 2007) describes research on RNA as the 21st century’s Big Bang in biology. The “stars” of the RNA universe are small regulatory RNAs known as microRNAs (miRNAs). These ∼22-nt-long RNAs were discovered in 1993 (Lee et al. 1993; Wightman et al. 1993), but their role in biology remained obscure until 2001, when the Tuschl, Ambros, and Bartel laboratories identified hundreds of miRNAs in different organisms (Lagos-Quintana et al. 2001; Lau et al. 2001; Lee and Ambros 2001). Functional analyses of these miRNAs and their associated complexes subsequently led to the discovery of RNA-guided genesilencing pathways as important novel principles in controlling gene expression. miRNAs indeed regulate the expression of a large proportion of eukaryotic protein-coding genes at the post-transcriptional level by sequence-specific binding to the 3 untranslated region (3 UTR) of mRNAs (Lim et al. 2005). To date, RNA silencing is known to play an important role in diverse biological phenomena such as development, stem cell maintenance, cell proliferation, survival, differentiation, metabolism, and cancer (Kloosterman and Plasterk 2006). The primary transcripts of miRNA genes are mainly processed by the nuclear RNase III enzyme Drosha into stem–loop-structured miRNA precursors (pre-miRNAs) (Lee et al. 2003; Ruby et al. 2007), which are transported via the Exportin-5 receptor to the cytoplasm (Yi et al. 2003). The cytoplasmic RNase III enzyme Dicer cleaves the pre-miRNAs into double-stranded (ds) RNAs of ∼22 nt (Bernstein et al. 2001; Hutvagner et al. 2001). One strand of these dsRNA intermediates associates as a mature miRNA with a member of the Argonaute protein family to generate an active ribonucleoprotein complex known as RNA-Induced Silencing Complex (RISC). Within this effector complex, both the miRNA and the Argonaute protein fulfill specific roles. The singlestranded miRNA recognizes the target mRNA with full or partial base-pair complementarity. The Argonaute protein helps the miRNA in searching for its target mRNA and, upon recognition, can either cleave it or remain tethered to the mRNA to repress its translation and/or regulate its stability. While the biochemistry of the miRNA-guided cleavage reactions is well understood (Martinez and Tuschl 2004; Schwarz et al. 2004), it is still controversial to what extent RISC interferes with the initiation or elongation of translation or controls mRNA stability by sequestration into cytoplasmic processing bodies (P-bodies) (Kiriakidou et al. 2007; Peters and Meister 2007; Pillai et al. 2007). The Argonaute (Ago) proteins can be subdivided into Ago-like and Piwi-like subfamilies. The mammalian Piwi-like proteins are specifically expressed during spermatogenesis, where they bind a novel set of small RNAs termed Piwi-interacting RNAs (piRNAs) (Aravin et al. 2006; Seto et al. 2007). In contrast, the Ago proteins are broadly expressed in somatic cells, associate with miRNAs and are key actors in different RNA silencing pathways (Peters and Meister 2007; Tolia and Joshua-Tor 2007). The Ago gene family consists of four (AGO1– AGO4) and five (Ago1–Ago5) members in human and mouse, respectively (Peters and Meister 2007). However, only the Ago2 protein displays endonucleolytic or “Slicer” activity and can therefore execute miRNA-directed cleavage of target mRNA, provided that the basepairing between the Ago2-associated miRNA and the mRNA sequence is perfect (Liu et al. 2004; Meister et al. 2004). In case of partial complementarity, the Ago2 protein fails to cleave, but instead interferes with translation of the target mRNA via its translational repression activity. In addition to Ago2, other mammalian Ago proteins are also part of miRNA effector complexes that mediate translational inhibition of target mRNAs (Liu et al. 2004; Meister et al. 2004). Gene disruption in the mouse demonstrated that the Ago2 protein is essential for embryonic development (Liu et al. 2004). To study the function of Ago2 in adult hematopoiesis, O’Carroll et al. (2007) have used conditional gene inactivation to bypass the embryonic lethality and report in this issue of Genes & Development that Ago2 is a key regulator of B-lymphoid and erythroid development. Retroviral rescue experiments surprisingly revealed that the Slicer endonuclease activity, which is a unique and defining feature of Ago2, is dispensable for hematopoietic developCorresponding author. E-MAIL busslinger@imp.ac.at; FAX (43/1) 79730-223150. Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1591407.

A Sequence-Based Model Accounts Largely for the Relationship of Intron Positions to Protein Structural Features

Claims of intron-structure correlations have played a major role in debates surrounding split gene origins. In the formative (as opposed to disruptive or "insertional") model of split gene origins, introns represent the scars of chimaeric gene assembly. When analyzed retrospectively, formative introns should tend to fall between modular units, if such units exist, or at least to exhibit a preference for sites favorable to chimaera formation. However, there is another possible source of preferences: under a disruptive model of split gene origins, fortuitous intron-structure correlations may arise because the gain of introns is biased with respect to flanking nucleotide sequences. To investigate the extent to which a sequence-biased intron gain model may account for the present-day distribution of introns, data on over 10,000 introns in eukaryotic protein-coding genes were integrated with structural data from a set of 1,851 nonredundant protein chains. The positions of introns with respect to secondary structures, solvent accessibility, and so-called "modules" were evaluated relative to the expectations of a null model, a disruptive model based on amino acid frequencies at splice junctions, and a formative model defined relative to these. The null model can be excluded for most structural features and is highly improbable when intron sites are grouped by reading frame phase. Phase-dependent correlations with secondary structure and side-chain surface accessibility are particularly strong. However, these phase-dependent correlations are explained largely by the sequence-based disruptive model.

Roles of the U5 snRNP in spliceosome dynamics and catalysis

Most protein-coding genes in eukaryotes are interrupted by non-coding intervening sequences (introns), which must be precisely removed from primary gene transcripts (pre-mRNAs) before translation of the message into protein. Intron removal by pre-mRNA splicing occurs in the nucleus and is catalysed by complex ribonucleoprotein machines called spliceosomes. These molecular machines consist of several small nuclear RNA molecules and their associated proteins [together termed snRNP (small nuclear ribonucleoprotein) particles], plus multiple accessory factors. Of particular interest are the U2, U5 and U6 snRNPs, which play crucial roles in the catalytic steps of splicing. In the present review, we summarize our current understanding of the role played by the protein components of the U5 snRNP in pre-mRNA splicing, which include some of the largest and most highly conserved nuclear proteins.

Preferential loss and gain of introns in 3′ portions of genes suggests a reverse-transcription mechanism of intron insertion

In an attempt to gain insight into the dynamics of intron evolution in eukaryotic protein-coding genes, the distributions of old introns, that are conserved between distant phylogenetic lineages, and new, lineage-specific introns along the gene length, were examined. A significant excess of old introns in 5′-regions of genes was detected. New introns, when analyzed in bulk, showed a nearly flat distribution from the 5′- to the 3′-end. However, analysis of new intron distributions in individual genomes revealed notable lineage-specific features. While in intron-poor genomes, particularly yeast Schizosaccharomyces pombe (Sp), the 5′-portions of genes contain a significantly greater number of new introns than the 3′-portions, the intron-rich genomes of humans and Arabidopsis show the opposite trend. These observations seem to be compatible with the view that introns are both lost and inserted in 3′-terminal portions of genes more often than in 5′-portions. Overrepresentation of 3′-terminal sequences among cDNAs that mediate intron loss appears to be the most likely explanation for the apparent preferential loss of introns in the distal parts of genes. Preferential insertion of introns in the 3′-portions suggests that introns might be inserted via a reverse-transcription-mediated pathway similar to that implicated in intron loss. This mechanism could involve duplication of a portion of the coding region during reverse transcription followed by homologous recombination and subsequent rapid sequence divergence in the copy that becomes a new intron.

The evolutionary gain of spliceosomal introns: sequence and phase preferences.

Theories regarding the evolution of spliceosomal introns differ in the extent to which the distribution of introns reflects either a formative role in the evolution of protein-coding genes or the adventitious gain of genetic elements. Here, systematic methods are used to assess the causes of the present-day distribution of introns in 10 families of eukaryotic protein-coding genes comprising 1,868 introns in 488 distinct alignment positions. The history of intron evolution inferred using a probabilistic model that allows ancestral inheritance of introns, gain of introns, and loss of introns reveals that the vast majority of introns in these eukaryotic gene families were not inherited from the most recent common ancestral genes, but were gained subsequently. Furthermore, among inferred events of intron gain that meet strict criteria of reliability, the distribution of sites of gain with respect to reading-frame phase shows a 5:3:2 ratio of phases 0, 1 and 2, respectively, and exhibits a nucleotide preference for MAG GT (positions -3 to +2 relative to the site of gain). The nucleotide preferences of intron gain may prove to be the ultimate cause for the phase bias. The phase bias of intron gain is sufficient to account quantitatively for the well-known 5:3:2 bias in phase frequencies among extant introns, a conclusion that holds even when taxonomic heterogeneity in phase patterns is considered. Thus, intron gain accounts for the vast majority of extant introns and for the bias toward phase 0 introns that previously was interpreted as evidence for ancient formative introns.

The RNA Polymerase II Core Promoter

The events leading to transcription of eukaryotic protein-coding genes culminate in the positioning of RNA polymerase II at the correct initiation site. The core promoter, which can extend ~35 bp upstream and/or downstream of this site, plays a central role in regulating initiation. Specific DNA elements within the core promoter bind the factors that nucleate the assembly of a functional preinitiation complex and integrate stimulatory and repressive signals from factors bound at distal sites. Although core promoter structure was originally thought to be invariant, a remarkable degree of diversity has become apparent. This article reviews the structural and functional diversity of the RNA polymerase II core promoter.

Genomewide analysis of mRNA processing in yeast using splicing-specific microarrays.

Introns interrupt almost every eukaryotic protein-coding gene, yet how the splicing apparatus interprets the genome during messenger RNA (mRNA) synthesis is poorly understood. We designed microarrays to distinguish spliced from unspliced RNA for each intron-containing yeast gene and measured genomewide effects on splicing caused by loss of 18 different mRNA processing factors. After accommodating changes in transcription and decay by using gene-specific indexes, functional relationships between mRNA processing factors can be identified through their common effects on spliced and unspliced RNA. Groups of genes with different dependencies on mRNA processing factors are also apparent. Quantitative polymerase chain reactions confirm the array-based finding that Prp17p and Prp18p are not dispensable for removal of introns with short branchpoint-to-3' splice site distances.

Multi-protein complexes in eukaryotic gene transcription.

Specific transcription initiation by RNA polymerase II at eukaryotic protein-coding genes involves the cooperative assembly at the core promoter of more than 40 distinct proteins--with a total mass of over 2 MDa--including RNA polymerase II itself and general/basal transcription initiation factors, to form a stable pre-initiation complex (PIC). In vivo, PIC assembly is a major point of regulation by sequence-specific transcription regulators (activators and repressors) and is hindered by the packaging of promoter DNA into nucleosomes and higher order chromatin structures. Genetic and biochemical studies have recently identified a variety of transcription cofactors/co-regulators (coactivators and corepressors) that interact with sequence-specific regulators and/or various components of the general/basal transcription machinery and are essential for regulated transcription. An emerging view from these studies is that regulators must target two types of transcription cofactors: chromatin-modifying/remodeling cofactors and general cofactors that associate with and/or influence the activities of components of the general/basal transcription machinery. The recent biochemical identification and characterization of many different chromatin-modifying and general transcription cofactors has revealed their often complex multi-subunit nature and a previously unsuspected level of structural and functional redundancy. Another emerging theme is the multi-functional nature of chromatin-modifying cofactor complexes that appear to couple gene-specific transcription to other cellular processes.

7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes.

The transcription of eukaryotic protein-coding genes involves complex regulation of RNA polymerase (Pol) II activity in response to physiological conditions and developmental cues. One element of this regulation involves phosphorylation of the carboxy-terminal domain (CTD) of the largest polymerase subunit by a transcription elongation factor, P-TEFb, which comprises the kinase CDK9 and cyclin T1 or T2 (ref. 1). Here we report that in human HeLa cells more than half of the P-TEFb is sequestered in larger complexes that also contain 7SK RNA, an abundant, small nuclear RNA (snRNA) of hitherto unknown function. P-TEFb and 7SK associate in a specific and reversible manner. In contrast to the smaller P-TEFb complexes, which have a high kinase activity, the larger 7SK/P-TEFb complexes show very weak kinase activity. Inhibition of cellular transcription by chemical agents or ultraviolet irradiation trigger the complete disruption of the P-TEFb/7SK complex, and enhance CDK9 activity. The transcription-dependent interaction of P-TEFb with 7SK may therefore contribute to an important feedback loop modulating the activity of RNA Pol II.

Rpb4, a Non-essential Subunit of Core RNA Polymerase II ofSaccharomyces cerevisiae Is Important for Activated Transcription of a Subset of Genes

A major role in the regulation of eukaryotic protein-coding genes is played by the gene-specific transcriptional regulators, which recruit the RNA polymerase II holoenzyme to the specific promoter. Several components of the mediator complex within the holoenzyme also have been shown to affect activation of different subsets of genes. Only recently has it been suggested that besides the largest subunit of RNA polymerase II, smaller subunits like Rpb3 and Rpb5 may have regulatory roles in expression of specific sets of genes. We report here, the role of Rpb4, a non-essential subunit of core RNA polymerase II, in activation of a subset of genes in Saccharomyces cerevisiae. We have shown below that whereas constitutive transcription is largely unaffected, activation from various promoters tested is severely compromised in the absence of RPB4. This activation defect can be rescued by the overexpression of cognate activators. We have localized the region of Rpb4 involved in activation to the C-terminal 24 amino acids. We have also shown here that transcriptional activation by artificial recruitment of the TATA-binding protein (TBP) to the promoter is also defective in the absence of RPB4. Surprisingly, the overexpression of RPB7 (the interacting partner of Rpb4) does not rescue the activation defect of all the promoters tested, although it rescues the activation defect of the heat shock element-containing promoter and the temperature sensitivity associated with RPB4 deletion. Overall, our results indicate that Rpb4 and Rpb7 play independent roles in transcriptional regulation of genes.

Widespread occurrence of spliceosomal introns in the rDNA genes of ascomycetes.

Spliceosomal (pre-mRNA) introns have previously been found in eukaryotic protein-coding genes, in the small nuclear RNAs of some fungi, and in the small- and large-subunit ribosomal DNA genes of a limited number of ascomycetes. How the majority of these introns originate remains an open question because few proven cases of recent and pervasive intron origin have been documented. We report here the widespread occurrence of spliceosomal introns (69 introns at 27 different sites) in the small- and large-subunit nuclear-encoded rDNA of lichen-forming and free-living members of the Ascomycota. Our analyses suggest that these spliceosomal introns are of relatively recent origin, i.e., within the Euascomycetes, and have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into rRNAs. The spliceosome itself, and not an external agent (e.g., transposable elements, group II introns), may have given rise to these introns. A nonrandom sequence pattern was found at sites flanking the rRNA spliceosomal introns. This pattern (AG-intron-G) closely resembles the proto-splice site (MAG-intron-R) postulated for intron insertions in pre-mRNA genes. The clustered positions of spliceosomal introns on secondary structures suggest that particular rRNA regions are preferred sites for insertion through reverse-splicing.

Structural Characterization of the Cysteine-rich Domain of TFIIH p44 Subunit

In an effort to understand the structure function relationship of TFIIH, a transcription/repair factor, we focused our attention on the p44 subunit, which plays a central role in both mechanisms. The amino-terminal portion of p44 has been shown to be involved in the regulation of the XPD helicase activity; here we show that its carboxyl-terminal domain is essential for TFIIH transcription activity and that it binds three zinc atoms through two independent modules. The first contains a C4 zinc finger motif, whereas the second is characterized by a CX(2)CX(2-4)FCADCD motif, corresponding to interleaved zinc binding sites. The solution structure of this second module reveals an unexpected homology with the regulatory domain of protein kinase C and provides a framework to study its role at the molecular level.

An Activator Target in the RNA Polymerase II Holoenzyme

Expression of protein-coding genes in eukaryotes involves the recruitment, by transcriptional activator proteins, of a transcription initiation apparatus consisting of greater than 50 polypeptides. Recent genetic and biochemical evidence in yeast suggests that a subset of these proteins, called SRB proteins, are likely targets for transcriptional activators. We demonstrate here, through affinity chromatography, photo-cross-linking, and surface plasmon resonance experiments, that the GAL4 activator interacts directly with the SRB4 subunit of the RNA polymerase II holoenzyme. The GAL4 activation domain binds to two essential segments of SRB4. The physiological relevance of this interaction is confirmed by mutations in SRB4, which occur within its GAL4-binding domain and which restore activation in vivo by a GAL4 derivative bearing a mutant activation domain.

Generality of a functional initiator consensus sequence

Transcriptional initiator (Inr) elements, like TATA boxes, are found in the core promoters of many eukaryotic protein-coding genes. To facilitate the dissection of transcription initiation mechanisms, and to identify Inr elements within known and newly-discovered genes, the precise DNA sequence requirements for Inr activity must be defined. Previously, we reported a preliminary Inr consensus sequence based on an extensive mutant analysis carried out in HeLa cell extracts. This analysis was limited, however, because it was performed only with an in vitro transcription assay and with the mutants in only one promoter context. In this study, we have assessed the general validity of the functional Inr consensus sequence by analyzing a selected set of mutants in additional promoter contexts and with both in vivo and in vitro assays. In addition, we analyzed the evolutionary conservation of the Inr consensus sequence by determining the sequence requirements for Inr function in Drosophila embryo extracts. The results demonstrate the generality and strict conservation among vertebrates and invertebrates of the Inr consensus sequence, Py Py A +1 N T/A Py Py.