Alternative Initiation Sites Research Articles

Dual CRALBP isoforms unveiled: iPSC-derived retinal modeling and AAV2/5-RLBP1 gene transfer raise considerations for effective therapy

Inherited retinal diseases (IRDs) are characterised by progressive vision loss. There are over 270 causative IRD genes and variants within the same gene can cause clinically distinct disorders. One example is RLBP1 that encodes CRALBP. CRALBP is an essential protein in the rod and cone visual cycles that take place primarily in the retinal pigment epithelium (RPE) but also in Müller cells of the neuroretina. RLBP1 variants lead to three clinical subtypes: Bothnia dystrophy, retinitis punctata albescens and Newfoundland rod-cone dystrophy. We modelled RLBP1-IRD subtypes using patient-specific iPSC-derived RPE and identified pathophysiological markers that served as pertinent therapeutic read-outs. We developed an AAV2/5-mediated gene supplementation strategy and performed a proof-of-concept study in the human models, which was validated in vivo in an Rlbp1-/- murine model. Most importantly, we identified a previously unsuspected smaller CRALBP isoform that is naturally and differentially expressed both in the human and murine retina. This previously unidentified isoform is produced from an alternative methionine initiation site. This work provides further insights into CRALBP expression and RLBP1-associated pathophysiology and raises important considerations for successful gene supplementation therapy.

A novel allelic donkey β-lactoglobulin I protein isoform generated by a non-AUG translation initiation codon is associated with a nonsynonymous SNP

β-Lactoglobulin I (β-LG I) is one of the most important whey proteins in donkey milk. However, to our knowledge, there has been no study focusing on the full nucleotide sequences of this gene (BLG I). Current investigation of donkey BLG I gene is very limited with only 2 variants (A and B) characterized so far at the protein level. Recently, a new β-LG I variant, with a significantly higher mass (+1,915 Da) than known variants has been detected. In this study, we report the whole nucleotide sequence of the BLG I gene from 2 donkeys, whose milk samples are characterized by the β-LG I SDS-PAGE band with a normal electrophoretic mobility (18,514.25 Da, β-LG I B1 form) the first, and by the presence of a unique β-LG I band with a higher electrophoretic mobility (20,428.5 Da, β-LG I D form) the latter. A high genetic variability was found all over the 2 sequenced BLG I alleles. In particular, 16 polymorphic sites were found in introns, one in the 5' flanking region, 3 SNPs in the 5' untranslated region and one SNP in the coding region (g.458G > A) located at the 40th nucleotide of exon 2 and responsible for the AA substitutions p.Asp28 > Asn in the mature protein. Two SNPs (g.920-922CAC > TGT and g.1871G/A) were genotyped in 93 donkeys of 2 Italian breeds (60 Ragusana and 33 Amiatina, respectively) and the overall frequencies of g.920-922CAC and g.1871A were 0.3065 and 0.043, respectively. Only the rare allele g.1871A was observed to be associated with the slower migrating β-LG I. Considering this genetic diversity and those found in the database, it was possible to deduce at least 5 different alleles (BLG I A, B, B1, C, D) responsible for 4 potential β-LG I translations. Among these alleles, B1 and D are those characterized in the present research, with the D allele of real novel identification. Haplotype data analysis suggests an evolutionary pathway of donkey BLG I gene and a possible phylogenetic map is proposed. Analyses of mRNA secondary structure showed relevant changes in the structures, as consequence of the g.1871G > A polymorphism, that might be responsible for the recognition of an alternative initiation site providing an additional signal peptide. The extension of 19 AA sequence to the mature protein, corresponding to the canonical signal peptide with an additional alanine residue, is sufficient to provide the observed molecular weight of the slower migrating β-LG I encoded by the BLG I D allele.

Modification of the second translation initiation site restricts the replication of foot-and-mouth disease virus in PK-15 cells

The translation initiation of foot-and-mouth disease virus (FMDV) occurs at two alternative initiation sites (Lab AUG and Lb AUG). Usually, the Lb AUG is more favorably used to initiate protein synthesis than the Lab AUG. To explore the effect of Lb AUG on FMDV replication and obtain FMDV with restricted replication, this initiation codon was mutated to a variety of non-AUG codons (UGG, AUC, CUG, and AAA). Fortunately, the modifications did not prevent viral viability but influenced replication characteristics of some FMDV mutants in a cell-specific manner, as was shown by the similar replication in BHK-21 cells and delayed growth kinetics in PK-15 cells. This attenuated phenotype of FMDV mutants in PK-15 cells was found to be correlated with reduced abilities to cleave eIF4GI and suppress interference (IFN) expression. As leader (L) protein was reported to be responsible for eIF4GI cleavage and inhibition of IFN expression, the in vivo L protein synthesis was examined during the infection of FMDV mutants. Our results showed that not only the total yield of L proteins was severely influenced but also the individual yield of L protein was seen to be affected, which implied that both the relative usage of the two initiation sites and overall translation efficiency were changed by Lb AUG modifications. In addition, the in vitro translation activity was also negatively regulated by Lb AUG mutations. Collectively, these findings suggested that the restricted replications of Lb AUG-modified FMDVs were related to the delayed eIF4GI cleavage and decreased ability to block IFN expression but were mainly determined by the inefficient translation initiation. FMDVs precisely with modifications of Lb AUG initiation codon may represent safer seed viruses for vaccine production.Key points• The polyprotein translation of FMDV initiates at two alternative initiation sites (Lab AUG and Lb AUG). In order to explore the effect of Lb AUG on FMDV replication and obtain FMDV with restricted replication, the Lb initiation AUG was mutated to a variety of non-AUG codons (UGG, AUC, CUG, and AAA), and four FMDV mutants with Lb AUG modification were generated.• We found that partial FMDV mutants grew almost as well as WT virus in BHK-21 cells, a typical cell line used for FMD vaccine production, but displayed impaired replication in IFN-competent PK-15 cells.• The attenuation of mutant FMDVs in PK-15 cells was found to be correlated with delayed eIF4GI cleavage and decreased ability to block IFN expression.• We proved that the attenuated phenotype of Lb AUG-modified FMDVs was mainly determined by the inefficient translation initiation, as demonstrated by the decrease of total yield of L proteins and individual production of L protein.• We successfully generated genetically engineered FMDV with attenuated phenotype. The approach of precise engineering of FMDV with the modification of initiation codon provides a safe platform to produce inactivated antigen vaccines.

Regulation of eIF2α by RNF4 Promotes Melanoma Tumorigenesis and Therapy Resistance

Among the hallmarks of melanoma are impaired proteostasis and rapid development of resistance to targeted therapy that represent a major clinical challenge. However, the molecular machinery that links these processes is unknown. Here we describe that by stabilizing key melanoma oncoproteins, the ubiquitin ligase RNF4 promotes tumorigenesis and confers resistance to targeted therapy in melanoma cells, xenograft mouse models, and patient samples. In patients, RNF4 protein and mRNA levels correlate with poor prognosis and with resistance to MAPK inhibitors. Remarkably, RNF4 tumorigenic properties, including therapy resistance, require the translation initiation factor initiation elongation factor alpha (eIF2α). RNF4 binds, ubiquitinates, and stabilizes the phosphorylated eIF2α (p-eIF2α) but not activating transcription factor 4 or C/EBP homologous protein that mediates the eIF2α-dependent integrated stress response. In accordance, p-eIF2α levels were significantly elevated in high-RNF4 patient-derived melanomas. Thus, RNF4 and p-eIF2α establish a positive feed-forward loop connecting oncogenic translation and ubiquitin-dependent protein stabilization in melanoma.

Adverse maternal environment and western diet impairs cognitive function and alters hippocampal glucocorticoid receptor promoter methylation in male mice.

Adverse maternal environment (AME) and high‐fat diet in early childhood increase the risk of cognitive impairment and depression later in life. Cognitive impairment associates with hippocampal dysfunction. A key regulator of hippocampal function is the glucocorticoid receptor. Increased hippocampal GR expression associates with cognitive impairment and depression. Transcriptional control of GR relies in part upon the DNA methylation status at multiple alternative initiation sites that are tissue specific, with exon 1.7 being hippocampal specific. Increased exon 1.7 expression associates with upregulated hippocampal GR expression in early life stress animal models. However, the effects of AME combined with postweaning western diet (WD) on offspring behaviors and the expression of GR exon 1 variants in the hippocampus are unknown. We hypothesized that AME and postweaning WD would impair cognitive function and cause depression‐like behavior in offspring in conjunction with dysregulated hippocampal expression of total GR and exon 1.7 variant in mice. We found that AME‐WD impaired learning and memory in male adult offspring concurrently with increased hippocampal expression of total GR and GR 1.7. We also found that increased GR 1.7 expression was associated with decreased DNA methylation at the GR 1.7 promoter. We speculate that decreased DNA methylation at the GR 1.7 promoter plays a role in AME‐WD induced increase of GR in the hippocampus. This increased GR expression may subsequently contribute to hippocampus dysfunction and lead to the cognitive impairment seen in this model.

Proteogenomic Approach to UTR Peptide Identification.

Recent sequencing technologies have highlighted translation of untranslated regions (UTRs) in genomes, although it remains unknown whether the translated products persist in a cell. Here, we propose a proteogenomic approach to UTR identification at the proteome level, which has been challenging due to the lack of corresponding sequences required for peptide spectrum matching. We address the challenge with constructing translated UTR (tUTR) database, consisting of all hypothetical sequences that can be translated from UTR by assuming non-AUG initiation at near-cognate start codons and stop codon readthrough. In the analysis of the H1299 cell line mass spectrometry (MS/MS) dataset, the tUTR DB-based proteogenomic approach enabled the detection of 52 5'-UTR and 9 3'-UTR peptides from 45 and 9 genes, respectively. The identified UTR peptides were validated via high spectral similarity with their synthetic peptides. The 5'-UTR peptides pointed out alternative initiation sites with non-AUG start codons, which exactly conformed to Kozak contexts of annotated initiation sites. It is also worth noting that our approach can detect translated amino acid sequences as well as provide evidence for UTR translation, while ribosome profiling provides only the translation evidence. For previously reported stop codon readthrough in MDH1 gene, we could confirm the amino acid inserted during the readthrough. Data are available via ProteomeXchange with identifier PXD016207.

Alternative Translation Initiation at a UUG Codon Gives Rise to Two Functional Variants of the Mitochondrial Protein Kgd4

Kgd4 is a novel subunit of the mitochondrial α-ketoglutarate dehydrogenase complex (KGDH). In yeast, the protein is present in two forms of unknown origin, as there is only one open reading frame and no alternative splicing. Here, we show that the two forms of Kgd4 derive from one mRNA that is translated by employing two alternative start sites. The standard, annotated AUG codon gives rise to the short form of the protein, while an upstream UUG codon is utilized to generate the larger form. However, both forms can be efficiently imported into mitochondria and stably incorporate into KGDH to support its activity. Translation of the long variant depends on sequences directly upstream of the alternative initiation site, demonstrating that translation initiation and its efficiency are dictated by the sequence context surrounding a specific codon. In summary, the two forms of Kgd4 follow a very unusual biogenesis pathway, supporting the notion that translation initiation in yeast is more flexible than it is widely recognized.

Alternative translation initiation generates the N-terminal truncated form of RUNX1 that retains hematopoietic activity

Transcription factor RUNX1 plays a crucial role in hematopoiesis and its activity is tightly regulated at both the transcriptional and posttranslational levels. However, translational control of RUNX1 expression has not been fully understood. In this study, we demonstrated that RUNX1b mRNA is translated from two alternative initiation sites, Met-1 and Met-25, giving full-length RUNX1b and a shorter protein lacking the first 24 amino acids (RUNX1ΔN24). Presence/absence of strong Kozak consensus sequences around Met-1 determines which initiation site is mainly used in RUNX1b cDNA. Selective disruption of either Met-1 or Met-25 abrogates expression of the corresponding protein while facilitating the production of another protein. The RUNX1b cDNA containing 65bp natural promoter sequences mainly produces full-length RUNX1b in human cord blood cells, but disruption of Met-1 in this cDNA also induced translation from Met-25. Consistent with these data, disruption of endogenous RUNX1b around Met-1 using CRISPR/Cas9 induced selective expression of RUNX1ΔΝ24 in several leukemia cell lines. RUNX1ΔN24 protein is more stable than full-length RUNX1b and retains hematopoietic activity. We also found that FLAG-tagged full-length RUNX1 showed altered activity, indicating the influence of N-terminal FLAG-tag on RUNX1 function. The alternative translation initiation of RUNX1b may participate in fine tuning RUNX1 activity.

Comprehensive profiling of translation initiation in influenza virus infected cells.

Translation can initiate at alternate, non-canonical start codons in response to stressful stimuli in mammalian cells. Recent studies suggest that viral infection and anti-viral responses alter sites of translation initiation, and in some cases, lead to production of novel immune epitopes. Here we systematically investigate the extent and impact of alternate translation initiation in cells infected with influenza virus. We perform evolutionary analyses that suggest selection against non-canonical initiation at CUG codons in influenza virus lineages that have adapted to mammalian hosts. We then use ribosome profiling with the initiation inhibitor lactimidomycin to experimentally delineate translation initiation sites in a human lung epithelial cell line infected with influenza virus. We identify several candidate sites of alternate initiation in influenza mRNAs, all of which occur at AUG codons that are downstream of canonical initiation codons. One of these candidate downstream start sites truncates 14 amino acids from the N-terminus of the N1 neuraminidase protein, resulting in loss of its cytoplasmic tail and a portion of the transmembrane domain. This truncated neuraminidase protein is expressed on the cell surface during influenza virus infection, is enzymatically active, and is conserved in most N1 viral lineages. We do not detect globally higher levels of alternate translation initiation on host transcripts upon influenza infection or during the anti-viral response, but the subset of host transcripts induced by the anti-viral response is enriched for alternate initiation sites. Together, our results systematically map the landscape of translation initiation during influenza virus infection, and shed light on the evolutionary forces shaping this landscape.

Single-probe RNA FISH in Yeast.

Quantitative profiling of mRNA expression is an important part of understanding the state of a cell. The technique of RNA Fluorescence In Situ Hybridization (FISH) involves targeting an RNA transcript with a set of 40 complementary fluorescently labeled DNA oligonucleotide probes. However, there are many circumstances such as transcripts shorter than 200 nt, splicing variations, or alternate initiation sites that create transcripts that would be indistinguishable to a set of multiple probes. To this end we adapted the standard FISH protocol to allow the use of a single probe with a single fluorophore to quantify the amount of transcripts inside budding yeast cells. In addition to allowing the quantification of short transcripts or short features of transcripts, this technique reduces the cost of performing FISH.

Abstract 1472: Alternate translation initiation regulation of PRDX5 mRNA by miR6855<->3p in basal<->like breast cancer cells

Abstract Human peroxiredoxins (PRDXs) are a superfamily of six thiol<->dependent peroxidases that are able to reduce hydrogen peroxide, alkyl hydroperoxides and peroxynitrite. Apart from the classical, apparently redundant function of mitochondrial and often peroxisomal peroxide neutralization shared by other peroxiredoxins, PRDX5 was initially discovered as a DNA binding transcriptional repressor regulating the biosynthesis of cytotoxic Alu RNAs through a mitochondrial<->localization signal<->truncated mature form (SPRDX5] of this protein that is accumulated in the nucleus and binds to a 60 bp nucleotide sequence at the Alu gene promoters. Our data suggest that SPRDX5 also binds to the Alu<->sequence<->containing BRCA2 gene silencer to prevent the binding of SLUG at the silencer thus enhancing the BRCA2 gene expression leading to the genotoxin and radiation<->resistance of aggressive SLUG<->high basal<->like breast cancer cells. PRDX5 mRNA contains two in<->frame start codons (AUGs) that are conditionally used as alternate translation initiation sites. Translation from the first AUG would result in the synthesis of a larger 214<->residue protein (LPRDX5) whereas the use of the second AUG would result in the production of a shorter 162<->residue polypeptide (SPRDX5). Since SPRDX5 lacks the mitochondrial localization signal, it is accumulated in the nucleus via its C<->terminal bipartite nuclear localization signal. We present evidence here that a miRNA, miR<->6855<->3p, binds the primary PRDX5 transcript between the two AUG codons and prevents the translation of the mRNA from the first AUG codon but not that from the second AUG codon thus favoring the initiation of translation from the second AUG codon. To characterize the effect of miR<->6855<->3p on Prdx5 mRNA translation, we developed three different PRDX5<->FLAG constructs in pCV3XFLAG14 (Sigma) that has Prdx5 ORF with or without mutation on the first or second AUG codon. Transfection of these constructs into BT549 cells followed by treatments with miR<->6855<->3p mimic or antagomiR and subcellular fractionation further supported our notion. We propose here that miR<->6855<->3p acts as a tsmiR by promoting the biosynthesis of the transcriptional regulator protein SPRDX5 which enhances the expression of the tumor suppressor protein BRCA2 leading to the genotoxin and radiation<->resistance of aggressive SLUG<->high basal<->like breast cancer cells. Supported in part by DOD-CDMRP IDEA Expansion Grant# BC103645 and NIH/NCI grant 1R21CA181920 01 to GC and 1U54RR026140 to SM. Citation Format: Gautam Chaudhuri, Smita Misra. Alternate translation initiation regulation of PRDX5 mRNA by miR6855<->3p in basal<->like breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1472. doi:10.1158/1538-7445.AM2017-1472

Primary and Metastatic Cutaneous Melanomas Express ALK Through Alternative Transcriptional Initiation.

A number of common driver mutations have been identified in melanoma, but other genetic or epigenetic aberrations are also likely to play a role in the pathogenesis of melanoma and present potential therapeutic targets. Translocations of the anaplastic lymphoma kinase (ALK), for example, have been reported in spitzoid melanocytic neoplasms leading to kinase-fusion proteins that result in immunohistochemically detectable ALK expression. In this study, we sought to determine whether ALK was also expressed in nonspitzoid primary and metastatic cutaneous melanomas. ALK immunohistochemistry was performed on 603 melanomas (303 primary and 300 metastatic tumors) from 600 patients. ALK immunohistochemistry expression was identified in 7 primary and 9 metastatic tumors. In 5 of 7 primary tumors and in 6 of 9 metastatic lesions, the majority of tumor cells were immunoreactive for ALK. In the other 2 primary and 3 metastatic lesions, positive staining was identified in less than half of the tumor cells. ALK positivity was found in the presence or absence of BRAF or NRAS mutations. In contrast to prior observations with ALK-positive Spitz tumors, none of the ALK-positive melanomas harbored a translocation. Instead, the ALK-positive melanomas predominantly expressed the recently described ALK isoform, ALK, which lacks the extracellular and transmembrane domains of wild-type ALK, consists primarily of the intracellular tyrosine kinase domain, and originates from an alternative transcriptional initiation site within the ALK gene. The findings are clinically relevant as patients with metastatic melanoma who have ALK expression may potentially benefit from treatment with ALK kinase inhibitors.

328. Proof-of-Principle Study Shows Efficient Skipping of Exon 2 Using Antisense Morpholino Oligomers

Currently, exon skipping therapies for Duchenne muscular dystrophy (DMD) have been developed for patients with out-of-frame deletions where treatment will lead translation of an internally truncated but partially functional dystrophin protein. In contrast, we are focusing on treating duplications mutations, accounting for around 6% of all mutations, resulting in wild-type transcript and a full-length protein. Modeling the most common single exon duplication we have developed the first duplication mouse containing a duplicated exon 2. We have performed a proof-of-principle study using antisense oligomer (AO)-induced exon 2 skipping using this Dup2 mouse. Intramuscular (TA) injections of exon 2-directed different antisense peptide-morpholino conjugates (PPMO) were performed at either 10 or 20 ug total PPMO (N=6 muscles each) doses. Mice were injected at 8 weeks and sacrificed 1 month later for muscle analysis of DMD mRNA and dystrophin protein expression. Additionally, systemic (tail vein) injections are being conducted of at a dose of 30 ug/kg, at 1 week, 2 week and 1 month timepoints. For the IM studies, a gradient of exon 2 exclusion was seen by RT-PCR at both doses with corresponding high levels of properly localized dystrophin protein by IF and western blot.Analyses of RT-PCR and protein expression are underway for the systemic delivery PPMOs. These data suggest that skipping of a duplicated exon 2 may be a feasible therapeutic approach, particularly because skipping of exon 2 may be associated with an apparently unlimited therapeutic window. Over-skipping - to the exclusion of exon 2 entirely - results in activation of an internal ribosome entry site (IRES) located in exon 5 of dystrophin that allows for cap-independent translation from an alternative initiation site within exon 6. This alternate dystrophin isoform is highly functional despite being N-truncated, consistent with the observation that patients expressing it have minimally symptomatic (or even asymptomatic) Becker muscular dystrophy (BMD), and suggesting a potential route to therapy for any of the approximately 5% of patients with mutations in the 5’ end of the gene.

Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA.

Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5′-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5′-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5′-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5′-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5′-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription.

505. Treatment of DMD 5’ Mutations Through Two Different exon2 Skipping Strategies: Intramuscular Delivery of rAAV9.snRNA Mediated Skipping and Antisense Morpholino Oligomers

To date, exon-skipping therapies for Duchenne muscular dystrophy (DMD) patients have focused on patients with out-of-frame exon deletions, in whom treatment results in larger but in-frame internal deletions which lead to translation of an internally truncated but partially functional dystrophin protein. We are developing exon-skipping therapies for exon duplication mutations, accounting for around 6% of all mutations, with the intent to induce production of wild-type transcripts and protein. As a model of the most common single exon duplication, we have developed a new mouse with a duplication of exon 2 (the Dup2 mouse) that largely recapitulates the findings in the standard mdx mouse. Using this mouse, we are testing both virally (AAV) mediated skipping induced by a modified U7snRNA (rAAV9.U7.ACCA), and antisense oligomer-induced skipping. Intramuscular injections of the tibialias anterior (TA) (N=6 muscles each) were performed rAAV9.U7.ACCA at 6 doses between 1×1010 and 1×1012 total vector genomes. Intramuscular (TA) injections of an exon 2-directed antisense peptide-morpholino conjugate (PPMO) were performed at doses of either 10 or 20 ug total PPMO (N=3 each). For each study, mice were sacrificed 1 month post injection and muscle analysis of DMD mRNA and dystrophin protein expression. Treatment with either modality results in significant skipping of the duplicated exon 2, and expression of a functional dystrophin isoform at levels of up to 30% of normal with AAV-mediated skipping. Physiology has been assessed in the AAV-treated mice, in which correction of TA absolute force deficits in comparison to the background Bl6 strain are seen, along with a partial yet significant correction of eccentric contraction injury in comparison to untreated Dup2 mice. These data suggest that skipping of a duplicated exon 2 may be a feasible therapeutic approach, particularly because skipping of exon 2 may be associated with an apparently unlimited therapeutic window. Over-skipping – to the exclusion of exon 2 entirely – results in activation of an internal ribosome entry site (IRES) located in exon 5 of dystrophin that allows for cap-independent translation from an alternative initiation site within exon 6. This alternate dystrophin isoform is highly functional despite being N-truncated, consistent with the observation that patients expressing it have minimally symptomatic (or even asymptomatic) Becker muscular dystrophy (BMD), and suggesting a potential route to therapy for any of the approximately 5% of patients with mutations in the 5’ end of the gene.

Exposing the subunit diversity and modularity of protein complexes by structural mass spectrometry approaches.

Although the number of protein-encoding genes in the human genome is only about 20000 not far from the amount found in the nematode worm genome, the number of proteins that are translated from these sequences is larger by several orders of magnitude. A number of mechanisms have evolved to enable this diversity. For example, genes can be alternatively spliced to create multiple transcripts; they may also be translated from different alternative initiation sites. After translation, hundreds of chemical modifications can be introduced in proteins, altering their chemical properties, folding, stability, and activity. The complexity is then further enhanced by the various combinations that are generated from the assembly of different subunit variants into protein complexes. This, in turn, confers structural and functional flexibility, and endows the cell with the ability to adapt to various environmental conditions. Therefore, exposing the variability of protein complexes is an important step toward understanding their biological functions. Revealing this enormous diversity, however, is not a simple task. In this review, we will focus on the array of MS-based strategies that are capable of performing this mission. We will also discuss the challenges that lie ahead, and the future directions toward which the field might be heading.

Quantitative analysis of mammalian translation initiation sites by FACS-seq.

An approach combining fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) was employed to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing AUG start codons. Using FACS-seq, we measured translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning the −6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon recognition and translation efficiency. However, dinucleotide interactions, which cannot be conveyed by a single motif, were also important for modeling TIS efficiency. Our dataset combined with modeling allowed us to predict genome-wide translation initiation efficiency for all mRNA transcripts. Additionally, we screened somatic TIS mutations associated with tumorigenesis to identify candidate driver mutations consistent with known tumor expression patterns. Finally, we implemented a quantitative leaky scanning model to predict alternative initiation sites that produce truncated protein isoforms and compared predictions with ribosome footprint profiling data. The comprehensive analysis of the TIS sequence space enables quantitative predictions of translation initiation based on genome sequence.

The mRNA of Human Cytoplasmic Arginyl-tRNA Synthetase Recruits Prokaryotic Ribosomes Independently

There are two isoforms of cytoplasmic arginyl-tRNA synthetase (hcArgRS) in human cells. The long form is a component of the multiple aminoacyl-tRNA synthetase complex, and the other is an N-terminal truncated form (NhcArgRS), free in the cytoplasm. It has been shown that the two forms of ArgRS arise from alternative translational initiation in a single mRNA. The short form is produced from the initiation at a downstream, in-frame AUG start codon. Interestingly, our data suggest that the alternative translational initiation of hcArgRS mRNA also takes place in Escherichia coli transformants. When the gene encoding full-length hcArgRS was overexpressed in E. coli, two forms of hcArgRS were observed. The N-terminal sequencing experiment identified that the short form was identical to the NhcArgRS in human cytoplasm. By constructing a bicistronic system, our data support that the mRNA encoding the N-terminal extension of hcArgRS has the capacity of independently recruiting E. coli ribosomes. Furthermore, two critical elements for recruiting prokaryotic ribosomes were identified, the “AGGA” core of the Shine-Dalgarno sequence and the “A-rich” sequence located just proximal to the alternative in-frame initiation site. Although the mechanisms of prokaryotic and eukaryotic translational initiation are distinct, they share some common features. The ability of the hcArgRS mRNA to recruit the prokaryotic ribosome may provide clues for shedding light on the mechanism of alternative translational initiation of hcArgRS mRNA in eukaryotic cells.

Characterization of three PDE5 isoforms in murine cardiomyocytes

Phosphodiesterase 5 (PDE5A) is responsible for hydrolysis of cGMP, a second messenger regulating many physiological functions in cardiac myocytes. PDE5A involvement in cardiac hypertrophy has been reported and the use of its inhibitor, sildenafil, has reverted the pathological increase of cardiac size in humans and in animal models (Nagendran et al., 2007). In humans, a single PDE5 gene encodes for three isoforms (PDE5A1, A2 and A3), which differ in their N-terminus being translated from alternative initiation sites (Lin et al., 2000). The isoforms exhibit specific tissue expression patterns and different sensitivities to pharmacological inhibitors. However, little is known about their specific biological roles. The existence of three murine PDE5A isoforms was predicted through human gene homology and confirmed by RT-PCR. Tissue expression pattern of each variant was uncovered by RT-PCR and western blot analysis. In adult heart, transcripts encoding for the three isoforms were detected. In cardiomyocytes primary cultures and cell lines PDE5A isoforms localization was revealed by fluorescence microscopy analysis and subcellular fractioning. Their phosphodiesterasic activities and sildenafil sensibilities were measured by radioactive assays. Finally, post-translational modifications were explored. Hypertrophic stimuli resulted in Ser 92 phosphorylation of PDE5A isoforms, possibly through by Protein Kinase A. In summary, the understanding of PDE5A isoforms localization and differential activation and activity might be an important step toward the improvement of the diagnostic, prognostic, and predictive values of PDE5A in hypertrophy treatment.

Expression Strategy of Aedes albopictus Densovirus

The transcription map of the Aedes albopictus densovirus (AalDNV) brevidensovirus was identified by Northern blotting, rapid amplification of cDNA ends (RACE) analysis, and RNase protection assays. AalDNV produced mRNAs of 3,359 (NS1), 3,345 (NS2), and 1,246 (VP) nucleotides. The two overlapping P7/7.4 NS promoters employed closely located alternate transcription initiation sites, positioned at either side of the NS1 initiation codon. All NS mRNAs coterminated with VP mRNA. All promoters, explored using luciferase assays, were functional in insect and human cell lines.