Influenza C virus CM2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence

The influenza C virus CM2 protein is a small glycosylated integral membrane protein (115 residues) that spans the membrane once and contains a cleavable signal sequence at its N terminus. The coding region for CM2 (CM2 ORF) is located at the C terminus of the 342-amino acid (aa) ORF of a colinear mRNA transcript derived from influenza C virus RNA segment 6. Splicing of the colinear transcript introduces a translational stop codon into the ORF and the spliced mRNA encodes the viral matrix protein (CM1) (242 aa). The mechanism of CM2 translation was investigated by using in vitro and in vivo translation of RNA transcripts. It was found that the colinear mRNA derived from influenza C virus RNA segment 6 serves as the mRNA for CM2. Furthermore, CM2 translation does not depend on any of the three in-frame methionine residues located at the beginning of CM2 ORF. Rather, CM2 is a proteolytic cleavage product of the p42 protein product encoded by the colinear mRNA: a cleavage event that involves the recognition and cleavage of an internal signal peptide presumably by signal peptidase resident in the endoplasmic reticulum. Alteration of the predicted signal peptidase cleavage site by mutagenesis blocked generation of CM2. The other polypeptide species resulting from the cleavage of p42, designated p31, contains the CM1 coding region and an additional C-terminal 17 aa (formerly the CM2 signal peptide). Protein p31, in comparison to CM1, displays characteristics of an integral membrane protein.

A curved RNA helix incorporating an internal loop with G·A and A·A non-Watson–Crick base pairing

The crystal structure of the RNA dodecamer 5′-GGCC(GAAA)GGCC-3′ has been determined from x-ray diffraction data to 2.3-Å resolution. In the crystal, these oligomers form double helices around twofold symmetry axes. Four consecutive non-Watson–Crick base pairs make up an internal loop in the middle of the duplex, including sheared G·A pairs and novel asymmetric A·A pairs. This internal loop sequence produces a significant curvature and narrowing of the double helix. The helix is curved by 34° from end to end and the diameter is narrowed by 24% in the internal loop. A Mn2+ ion is bound directly to the N7 of the first guanine in the Watson–Crick region following the internal loop and the phosphate of the preceding residue. This Mn2+ location corresponds to a metal binding site observed in the hammerhead catalytic RNA.

Two functionally distinct subsites for the binding of internal blockers to the pore of voltage-activated K+ channels

Many blockers of Na+ and K+ channels act by blocking the pore from the intracellular side. For Shaker K+ channels, such intracellular blockers vary in their functional effect on slow (C-type) inactivation: Some blockers interfere with C-type inactivation, whereas others do not. These functional differences can be explained by supposing that there are two overlapping “subsites” for blocker binding, only one of which inhibits C-type inactivation through an allosteric effect. We find that the ability to bind to these subsites depends on specific structural characteristics of the blockers, and correlates with the effect of mutations in two distinct regions of the channel protein. These interactions are important because they affect the ability of blockers to produce use-dependent inhibition.

Limited internal friction in the rate-limiting step of a two-state protein folding reaction

Small, single-domain proteins typically fold via a compact transition-state ensemble in a process well fitted by a simple, two-state model. To characterize the rate-limiting conformational changes that underlie two-state folding, we have investigated experimentally the effects of changing solvent viscosity on the refolding of the IgG binding domain of protein L. In conjunction with numerical simulations, our results indicate that the rate-limiting conformational changes of the folding of this domain are strongly coupled to solvent viscosity and lack any significant “internal friction” arising from intrachain collisions. When compared with the previously determined solvent viscosity dependencies of other, more restricted conformational changes, our results suggest that the rate-limiting folding transition involves conformational fluctuations that displace considerable amounts of solvent. Reconciling evidence that the folding transition state ensemble is comprised of highly collapsed species with these and similar, previously reported results should provide a significant constraint for theoretical models of the folding process.

Perceptual learning reflects external noise filtering and internal noise reduction through channel reweighting

To investigate the nature of plasticity in the adult visual system, perceptual learning was measured in a peripheral orientation discrimination task with systematically varying amounts of external (environmental) noise. The signal contrasts required to achieve threshold were reduced by a factor or two or more after training at all levels of external noise. The strong quantitative regularities revealed by this novel paradigm ruled out changes in multiplicative internal noise, changes in transducer nonlinearites, and simple attentional tradeoffs. Instead, the regularities specify the mechanisms of perceptual learning at the behavioral level as a combination of external noise exclusion and stimulus enhancement via additive internal noise reduction. The findings also constrain the neural architecture of perceptual learning. Plasticity in the weights between basic visual channels and decision is sufficient to account for perceptual learning without requiring the retuning of visual mechanisms.

PDZ Motifs in PTP-BL and RIL Bind to Internal Protein Segments in the LIM Domain Protein RIL

The specificity of protein–protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells.

Dissection of De Novo Membrane Insertion Activities of Internal Transmembrane Segments of ATP-Binding-Cassette Transporters: Toward Understanding Topological Rules for Membrane Assembly of Polytopic Membrane Proteins

The membrane assembly of polytopic membrane proteins is a complicated process. Using Chinese hamster P-glycoprotein (Pgp) as a model protein, we investigated this process previously and found that Pgp expresses more than one topology. One of the variations occurs at the transmembrane (TM) domain including TM3 and TM4: TM4 inserts into membranes in an Nin-Cout rather than the predicted Nout-Cin orientation, and TM3 is in cytoplasm rather than the predicted Nin-Cout orientation in the membrane. It is possible that TM4 has a strong activity to initiate the Nin-Cout membrane insertion, leaving TM3 out of the membrane. Here, we tested this hypothesis by expressing TM3 and TM4 in isolated conditions. Our results show that TM3 of Pgp does not have de novo Nin-Cout membrane insertion activity whereas TM4 initiates the Nin-Cout membrane insertion regardless of the presence of TM3. In contrast, TM3 and TM4 of another polytopic membrane protein, cystic fibrosis transmembrane conductance regulator (CFTR), have a similar level of de novo Nin-Cout membrane insertion activity and TM4 of CFTR functions only as a stop-transfer sequence in the presence of TM3. Based on these findings, we propose that 1) the membrane insertion of TM3 and TM4 of Pgp does not follow the sequential model, which predicts that TM3 initiates Nin-Cout membrane insertion whereas TM4 stops the insertion event; and 2) “leaving one TM segment out of the membrane” may be an important folding mechanism for polytopic membrane proteins, and it is regulated by the Nin-Cout membrane insertion activities of the TM segments.

A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity

This study addresses the properties of a newly identified internal ribosome entry site (IRES) contained within the mRNA of the homeodomain protein Gtx. Sequential deletions of the 5′ untranslated region (UTR) from either end did not define distinct IRES boundaries; when five nonoverlapping UTR fragments were tested, four had IRES activity. These observations are consistent with other cellular IRES analyses suggesting that some cellular IRESes are composed of segments (IRES modules) that independently and combinatorially contribute to overall IRES activity. We characterize a 9-nt IRES module from the Gtx 5′ UTR that is 100% complementary to the 18S rRNA at nucleotides 1132–1124. In previous work, we demonstrated that this mRNA segment could be crosslinked to its complement within intact 40S subunits. Here we show that increasing the number of copies of this IRES module in the intercistronic region of a dicistronic mRNA strongly enhances IRES activity in various cell lines. Ten linked copies increased IRES activity up to 570-fold in Neuro 2a cells. This level of IRES activity is up to 63-fold greater than that obtained by using the well characterized encephalomyocarditis virus IRES when tested in the same assay system. When the number of nucleotides between two of the 9-nt Gtx IRES modules was increased, the synergy between them decreased. In light of these findings, we discuss possible mechanisms of ribosome recruitment by cellular mRNAs, address the proposed role of higher order RNA structures on cellular IRES activity, and suggest parallels between IRES modules and transcriptional enhancer elements.

Identification of eIF2Bγ and eIF2γ as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach

The 5′-untranslated region of hepatitis C virus (HCV) is highly conserved, folds into a complex secondary structure, and functions as an internal ribosome entry site (IRES) to initiate translation of HCV proteins. We have developed a selection system based on a randomized hairpin ribozyme gene library to identify cellular factors involved in HCV IRES function. A retroviral vector ribozyme library with randomized target recognition sequences was introduced into HeLa cells, stably expressing a bicistronic construct encoding the hygromycin B phosphotransferase gene and the herpes simplex virus thymidine kinase gene (HSV-tk). Translation of the HSV-tk gene was mediated by the HCV IRES. Cells expressing ribozymes that inhibit HCV IRES-mediated translation of HSV-tk were selected via their resistance to both ganciclovir and hygromycin B. Two ribozymes reproducibly conferred the ganciclovir-resistant phenotype and were shown to inhibit IRES-mediated translation of HCV core protein but did not inhibit cap-dependent protein translation or cell growth. The functional targets of these ribozymes were identified as the gamma subunits of human eukaryotic initiation factors 2B (eIF2Bγ) and 2 (eIF2γ), respectively. The involvement of eIF2Bγ and eIF2γ in HCV IRES-mediated translation was further validated by ribozymes directed against additional sites within the mRNAs of these genes. In addition to leading to the identification of cellular IRES cofactors, ribozymes obtained from this cellular selection system could be directly used to specifically inhibit HCV viral translation, thereby facilitating the development of new antiviral strategies for HCV infection.

Identification of two short internal ribosome entry sites selected from libraries of random oligonucleotides

Sequences that control translation of mRNA may play critical roles in regulating protein levels. One such element is the internal ribosome entry site (IRES). We previously showed that a 9-nt segment in the 5′ leader sequence of the mRNA encoding Gtx homeodomain protein could function as an IRES. To identify other short sequences with similar properties, we designed a selection procedure that uses a retroviral vector to express dicistronic mRNAs encoding enhanced green and cyan fluorescent proteins as the first and second cistrons, respectively. Expression of the second cistron was dependent upon the intercistronic sequences and was indicative of IRES activity. B104 cells were infected with two retroviral libraries that contained random sequences of 9 or 18 nt in the intercistronic region. Cells expressing both cistrons were sorted, and sequences recovered from selected cells were reassayed for IRES activity in a dual luciferase dicistronic mRNA. Two novel IRESes were identified by this procedure, and both contained segments with complementarity to 18S rRNA. When multiple copies of either segment were linked together, IRES activities were dramatically enhanced. Moreover, these synthetic IRESes were differentially active in various cell types. These properties are similar to those of the previously identified 9-nt IRES module from Gtx mRNA. These results provide further evidence that short nucleotide sequences can function as IRESes and support the idea that some cellular IRESes may be composed of shorter functional modules. The ability to identify IRES modules with specific expression properties may be useful in the design of vectors for biotechnology and gene therapy.

Transcript leader regions of two Saccharomyces cerevisiae mRNAs contain internal ribosome entry sites that function in living cells

In higher eukaryotes, translation of some mRNAs occurs by internal initiation. It is not known, however, whether this mechanism is used to initiate the translation of any yeast mRNAs. In this report, we identify naturally occurring nucleotide sequences that function as internal ribosome entry sites (IRESes) within the 5′ leader sequences of Saccharomyces cerevisiae YAP1 and p150 mRNAs. When tested in the 5′ untranslated regions of monocistronic reporter genes, both leader sequences enhanced translation efficiency in vegetatively growing yeast cells. Moreover, when tested in the intercistronic region of dicistronic mRNAs, both sequences were shown to contain IRESes that functioned in living cells. The activity of the p150 leader was much greater than that of the YAP1 leader. The second cistron was not expressed in control dicistronic constructs that lacked these sequences or contained the 5′ leader sequence of the CLN3 mRNA in the intercistronic region. Further analyses of the p150 IRES revealed that it contained several nonoverlapping segments that were able independently to mediate internal initiation. These results suggested a modular composition for the p150 IRES that resembled the composition of IRESes contained within some cellular mRNAs of higher eukaryotes. Both YAP1 and p150 leaders contain several complementary sequence matches to yeast 18S rRNA. The findings are discussed in terms of our understanding of internal initiation in higher eukaryotes.

Cis-acting requirements in flanking DNA for the programmed elimination of mse2.9: a common mechanism for deletion of internal eliminated sequences from the developing macronucleus of Tetrahymena thermophila

During macronuclear development in the ciliated protozoan Tetrahymena thermophila, extensive DNA deletions occur, eliminating thousands of internal eliminated sequences (IESs). Using an rDNA-based transformation assay we have analyzed the role during DNA deletion of DNA flanking mse2.9, an IES within the second intron of a gene encoding an as yet incompletely characterized protein. We establish that a cis-acting sequence for mse2.9 deletion acts at a distance to specify deletion boundaries. A complex sequence element necessary for efficient and accurate mse2.9 deletion is located in the region 47–81 bp from the right side of mse2.9. The ability of a variety of IES flanking sequences to rescue a processing deficient mse2.9 construct indicates that some cis-acting signal is shared among different IESs. In addition, the short intronic sequence that flanks mse2.9 is able to direct efficient and accurate processing. Despite no obvious sequence similarity between mse2.9 and other IESs, we suggest that a common mechanism is used to delete different families of IESs in Tetrahymena.

Efficient construction of long DNA duplexes with internal non-Watson–Crick motifs and modifications

We have developed a semi-synthetic approach for preparing long stretches of DNA (>100 bp) containing internal chemical modifications and/or non-Watson–Crick structural motifs which relies on splint-free, cell-free DNA ligations and recycling of side-products by non-PCR thermal cycling. A double-stranded DNA PCR fragment containing a polylinker in its middle is digested with two restriction enzymes and a small insert (∼20 bp) containing the modification or non-Watson–Crick motif of interest is introduced into the middle. Incorrect products are recycled to starting materials by digestion with appropriate restriction enzymes, while the correct product is resistant to digestion since it does not contain these restriction sites. This semi-synthetic approach offers several advantages over DNA splint-mediated ligations, including fewer steps, substantially higher yields (∼60% overall yield) and ease of use. This method has numerous potential applications, including the introduction of modifications such as fluorophores and cross-linking agents into DNA, controlling the shape of DNA on a large scale and the study of non-sequence-specific nucleic acid–protein interactions.

Internal initiation of translation of five dendritically localized neuronal mRNAs

In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5′ leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the α subunit of calcium–calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5′ untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and α-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5′ untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overexpression of a protein that binds the cap-binding protein eIF4E, it was revealed that translation of these mRNAs had both cap-dependent and cap-independent components. The cap-independent component was further analyzed by inserting the 5′ leader sequences into the intercistronic region of dicistronic mRNAs. All five leader sequences mediated internal initiation via internal ribosome entry sites (IRESes). The RC3 IRES was most active and was further characterized after transfection in primary neurons. Although translation mediated by this IRES occurred throughout the cell, it was relatively more efficient in dendrites. These data suggest that IRESes may increase translation efficiency at postsynaptic sites after synaptic activation.