Untranslated Region Region Research Articles

Comparative Analysis of HPV16 Variants in the Untranslated Regulatory Region, L1, and E6 Genes among Vaccinated and Unvaccinated Young Women: Assessing Vaccine Efficacy and Viral Diversity.

HPV16 is occasionally detected in vaccinated women who received the bivalent HPV16/18 vaccine, usually at low viral loads. This study explored potential differences in HPV16 variants between vaccinated and unvaccinated women. HPV16-postive viral loads were detected in 1.9% (17/875) and 13% (162/760) of vaccinated and unvaccinated women, respectively, showcasing the vaccine's high efficacy. The L1, E6, and URR regions of HPV16 were sequenced from genital swabs from 16 vaccinated and 25 unvaccinated women in the HAVANA (HPV Among Vaccinated And Non-vaccinated Adolescents) study. The majority of HPV16 variants from vaccinated and unvaccinated women clustered similarly with sub-lineages A1 and A2. Additionally, a separate cluster within lineage A was found, with the variants sharing the L1-located SNP A753G (synonymous) and the URR-located SNP T340C, which did not occur in the other variants. Furthermore, four variants from vaccinated women had relatively long branches, but were not characterized by specific SNPs. The frequency of G712A in the URR was the only SNP observed to be marginally higher among vaccinated women than unvaccinated women. Non-synonymous SNPs T266A in the FG-loop of L1 and L83V in E6 were common among variants from vaccinated and unvaccinated women, but present in similar frequencies. In conclusion, the detection of HPV16 in vaccinated (and unvaccinated) women seemed to be the result of random circulation within this study population.

Molecular cloning and characterization of FLOWERING LOCUS T (FT)gene from kinnow mandarin (Citrus reticulata)

FLOWERING LOCUS T (FT) is considered as a crucial regulator involved in early flowering induction which functions by switching the vegetative phase into reproductive phase. The role of early flowering has been elucidated by many workers but till date, there has been no report on cloning of FT gene from Kinnow mandarin (Citrus reticulata Blanco). An experiment was conducted during 2022–23 at Punjab Agricultural University, Ludhiana, Punjab for molecular cloning and characterization of FT gene from Kinnow mandarin. During the study, FT gene was cloned into pGEM-T Easy vector and characterized using in silico analysis. Sequence analysis revealed that the FT gene is comprised of 822 nucleotides, including a 5’- untranslated region (UTR) of 137 bp, a coding sequence (CDS) of 534 bp, and a 3’-UTR region of 151 bp. The BLASTn results of CrFT CDS showed high similarity to FT-like genes of other citrus species including C. unshiu, C. sinensis, and C. clementina which were found homologous to Phosphatidylethanolamine binding protein (PEBP). The predicted molecular weight of protein was approximately 20 kDa and isoelectric point (pI) was 6.73 indicating slightly acidic nature of the protein. The instability, aliphatic and GRAVY (Grand Average of Hydropathy) indices of the protein were determined as 40.97, 73.16 and -0.491, respectively. This study would help to elucidate the molecular mechanism behind flowering in Kinnow mandarin and facilitate the molecular breeding studies targeted to obtain early flowering phenotypes.

A Fur family protein BosR is a novel RNA-binding protein that controls rpoS RNA stability in the Lyme disease pathogen.

The σ54-σS sigma factor cascade plays a central role in regulating differential gene expression during the enzootic cycle of Borreliella burgdorferi, the Lyme disease pathogen. In this pathway, the primary transcription of rpoS (which encodes σS) is under the control of σ54 which is activated by a bacterial enhancer-binding protein (EBP), Rrp2. The σ54-dependent activation in B. burgdorferi has long been thought to be unique, requiring an additional factor, BosR, a homologue of classical Fur/PerR repressor/activator. However, how BosR is involved in this σ54-dependent activation remains unclear and perplexing. In this study, we demonstrate that BosR does not function as a regulator for rpoS transcriptional activation. Instead, it functions as a novel RNA-binding protein that governs the turnover rate of rpoS mRNA. We further show that BosR directly binds to the 5' untranslated region (UTR) of rpoS mRNA, and the binding region overlaps with a region required for rpoS mRNA degradation. Mutations within this 5'UTR region result in BosR-independent RpoS production. Collectively, these results uncover a novel role of Fur/PerR family regulators as RNA-binding proteins and redefine the paradigm of the σ54-σS pathway in B. burgdorferi.

N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA.

Epitranscriptomic RNA modifications can regulate the stability of mRNA and affect cellular and viral RNA functions. The N4-acetylcytidine (ac4C) modification in the RNA viral genome was recently found to promote viral replication; however, the mechanism by which RNA acetylation in the host mRNA regulates viral replication remains unclear. To help elucidate this mechanism, the roles of N-acetyltransferase 10 (NAT10) and ac4C during the infection and replication processes of the alphavirus, Sindbis virus (SINV), were investigated. Cellular NAT10 was upregulated, and ac4C modifications were promoted after alphavirus infection, while the loss of NAT10 or inhibition of its N-acetyltransferase activity reduced alphavirus replication. The NAT10 enhanced alphavirus replication as it helped to maintain the stability of lymphocyte antigen six family member E mRNA, which is a multifunctional interferon-stimulated gene that promotes alphavirus replication. The ac4C modification was thus found to have a non-conventional role in the virus life cycle through regulating host mRNA stability instead of viral mRNA, and its inhibition could be a potential target in the development of new alphavirus antivirals.IMPORTANCEThe role of N4-acetylcytidine (ac4C) modification in host mRNA and virus replication is not yet fully understood. In this study, the role of ac4C in the regulation of Sindbis virus (SINV), a prototype alphavirus infection, was investigated. SINV infection results in increased levels of N-acetyltransferase 10 (NAT10) and increases the ac4C modification level of cellular RNA. The NAT10 was found to positively regulate SINV infection in an N-acetyltransferase activity-dependent manner. Mechanistically, the NAT10 modifies lymphocyte antigen six family member E (LY6E) mRNA-the ac4C modification site within the 3'-untranslated region (UTR) of LY6E mRNA, which is essential for its translation and stability. The findings of this study demonstrate that NAT10 regulated mRNA stability and translation efficiency not only through the 5'-UTR or coding sequence but also via the 3'-UTR region. The ac4C modification of host mRNA stability instead of viral mRNA impacting the viral life cycle was thus identified, indicating that the inhibition of ac4C could be a potential target when developing alphavirus antivirals.

3'UTR heterogeneity and cancer progression.

The majority of human mRNAs generate alternative 3' untranslated regions (UTRs) through various processes, including RNA modifications such as RNA editing, m6A methylation, and alternative polyadenylation (APA), with 3'UTR splicing as an emerging mechanism. Multiple factors, ranging from the genome to transcriptome level, regulate these processes and contribute to 3'UTR heterogeneity. Genomic variants in 3'UTR regions as well as aberrant 3'UTR processing alter the transcriptomic landscape and are associated with cancer. Increasing evidence, aided by high-resolution sequencing technologies and large-scale computational analyses, points towards potential crosstalk between these processes, whose deregulation may further contribute to cancer pathogenesis. In-depth characterization of these events will increase our appreciation of their significance and help to drive therapeutic development in this field.

A novel approach to analyze the association characteristics between post-spliced introns and their corresponding mRNA.

Studies have shown that post-spliced introns promote cell survival when nutrients are scarce, and intron loss/gain can influence many stages of mRNA metabolism. However, few approaches are currently available to study the correlation between intron sequences and their corresponding mature mRNA sequences. Here, based on the results of the improved Smith-Waterman local alignment-based algorithm method (SW method) and binding free energy weighted local alignment algorithm method (BFE method), the optimal matched segments between introns and their corresponding mature mRNAs in Caenorhabditis elegans (C.elegans) and their relative matching frequency (RF) distributions were obtained. The results showed that although the distributions of relative matching frequencies on mRNAs obtained by the BFE method were similar to the SW method, the interaction intensity in 5'and 3'untranslated regions (UTRs) regions was weaker than the SW method. The RF distributions in the exon-exon junction regions were comparable, the effects of long and short introns on mRNA and on the five functional sites with BFE method were similar to the SW method. However, the interaction intensity in 5'and 3'UTR regions with BFE method was weaker than with SW method. Although the matching rate and length distribution shape of the optimal matched fragment were consistent with the SW method, an increase in length was observed. The matching rates and the length of the optimal matched fragments were mainly in the range of 60%-80% and 20-30bp, respectively. Although we found that there were still matching preferences in the 5'and 3'UTR regions of the mRNAs with BFE, the matching intensities were significantly lower than the matching intensities between introns and their corresponding mRNAs with SW method. Overall, our findings suggest that the interaction between introns and mRNAs results from synergism among different types of sequences during the evolutionary process.

Whole-genome sequencing of foot-and-mouth disease virus serotype O/PanAsia-2/QOM-15 and comparison of its VP1-encoding region with two vaccine strains.

Despite widespread vaccination against foot-and-mouth disease, many outbreaks still occur in endemic areas. We attempted to determine the genetic and antigenic properties of the O/PanAsia-2/QOM-15 foot-and-mouth disease virus new vaccine strain. Thus, whole-genome sequencing was used to identify vulnerable pinpoint sites across the genome. The VP1 sequence (1D gene) of the O/PanAsia-2/QOM-15 viral genome was then compared to the VP1 sequences of two previously used vaccine strains, O/PanAsia (JQ321837) and O/PanAsia-2 (JN676146). The antigenic relationship of these three viruses was calculated by the two dimensional-virus neutralization test. At the nucleotide level, 47 single variants were identified, of which 19.00% were in the 5' untranslated region (UTR), 79.00% in the polyprotein region, and 2.00% in the 3' UTR region. Approximately half of the single nucleotide polymorphisms that have occurred in 1D gene resulted in amino acid (AA) substitutions in the VP1 structure. The single nucleotide polymorphisms also caused AA substitutions in other structural proteins, including VP2 and VP3, and some non-structural proteins (Lpro, 2C, and 3A). The O/PanAsia-2/QOM-15 shared higher sequence similarity with O/PanAsia-2 (91.00%) compared to O/PanAsia (87.30%). Evaluating r-value showed that the antigenic relationship of O/PanAsia-2/QOM-15 with O/PanAsia-2 (29.00%) was greater than that of the O/PanAsia (24.00%); however, all three viruses were immunologically distinct. After 10 years, the alteration of virus antigenicity and the lack of detectable adaptive pressure on VP1 sequence suggest that studying genetic dynamics beyond the VP1 region is necessary to evaluate FMDV pathogenicity and vaccine failure.

Targeted Sequencing of Cytokine-Induced PI3K-Related Genes in Ulcerative Colitis, Colorectal Cancer and Colitis-Associated Cancer.

Chronic relapsing inflammatory bowel disease is strongly linked to an increased risk of colitis-associated cancer (CAC). One of the well-known inflammatory carcinogenesis pathways, phosphatidylinositol 3-kinase (PI3K), was identified to be a crucial mechanism in long-standing ulcerative colitis (UC). The goal of this study was to identify somatic variants in the cytokine-induced PI3K-related genes in UC, colorectal cancer (CRC) and CAC. Thirty biopsies (n = 8 long-standing UC, n = 11 CRC, n = 8 paired normal colorectal mucosa and n = 3 CAC) were subjected to targeted sequencing on 13 PI3K-related genes using Illumina sequencing and the SureSelectXT Target Enrichment System. The Genome Analysis Toolkit was used to analyze variants, while ANNOVAR was employed to detect annotations. There were 5116 intronic, 355 exonic, 172 untranslated region (UTR) and 59 noncoding intronic variations detected across all samples. Apart from a very small number of frameshifts, the distribution of missense and synonymous variants was almost equal. We discovered changed levels of IL23R, IL12Rß1, IL12Rß2, TYK2, JAK2 and OSMR in more than 50% of the samples. The IL23R variant in the UTR region, rs10889677, was identified to be a possible variant that might potentially connect CAC with UC and CRC. Additional secondary structure prediction using RNAfold revealed that mutant structures were more unstable than wildtype structures. Further functional research on the potential variants is, therefore, highly recommended since it may provide insight on the relationship between inflammation and cancer risk in the cytokine-induced PI3K pathway.

Genetic polymorphisms of PKLR gene and their associations with milk production traits in Chinese Holstein cows.

Our previous work had confirmed that pyruvate kinase L/R (PKLR) gene was expressed differently in different lactation periods of dairy cattle, and participated in lipid metabolism through insulin, PI3K-Akt, MAPK, AMPK, mTOR, and PPAR signaling pathways, suggesting that PKLR is a candidate gene to affect milk production traits in dairy cattle. Here, we verified whether this gene has significant genetic association with milk yield and composition traits in a Chinese Holstein cow population. In total, we identified 21 single nucleotide polymorphisms (SNPs) by resequencing the entire coding region and partial flanking region of PKLR gene, in which, two SNPs were located in 5′ promoter region, two in 5′ untranslated region (UTR), three in introns, five in exons, six in 3′ UTR and three in 3′ flanking region. The single marker association analysis displayed that all SNPs were significantly associated with milk yield, fat and protein yields or protein percentage (p ≤ 0.0497). The haplotype block containing all the SNPs, predicted by Haploview, had a significant association with fat yield and protein percentage (p ≤ 0.0145). Further, four SNPs in 5′ regulatory region and eight SNPs in UTR and exon regions were predicted to change the transcription factor binding sites (TFBSs) and mRNA secondary structure, respectively, thus affecting the expression of PKLR, leading to changes in milk production phenotypes, suggesting that these SNPs might be the potential functional mutations for milk production traits in dairy cattle. In conclusion, we demonstrated that PKLR had significant genetic effects on milk production traits, and the SNPs with significant genetic effects could be used as candidate genetic markers for genomic selection (GS) in dairy cattle.

Characterization of N6-methyladenosine in cattle-yak testis tissue.

N6-methyladenosine (m6A) is the most common form of eukaryotic mRNA modification, and it has been shown to exhibit broad regulatory activity in yeast, plants, and mammals. The specific role of m6A methylation as a regulator of spermatogenesis, however, has yet to be established. In this experiment, through a series of preliminary studies and methylated RNA immunoprecipitation sequencing, the m6A map of cattle-yak testicular tissue was established as a means of exploring how m6A modification affects cattle-yak male infertility. Cattle-yak testis tissues used in this study were found to contain sertoli cells and spermatogonia. Relative to sexually mature yak samples, those isolated from cattle-yak testis exhibited slightly reduced levels of overall methylation, although these levels were significantly higher than those in samples from pre-sexually mature yaks. Annotation analyses revealed that differentially methylated peaks were most concentrated in exonic regions, with progressively lower levels of concentration in the 3'-untranslated region (UTR) and 5'-UTR regions. To further explore the role of such m6A modification, enrichment analyses were performed on differentially methylated and differentially expressed genes in these samples. For the cattle-yaks vs. 18-months-old yaks group comparisons, differentially methylated genes were found to be associated with spermatogenesis-related GO terms related to the cytoskeleton and actin-binding, as well as with KEGG terms related to the regulation of the actin cytoskeleton and the MAPK signaling pathway. Similarly, enrichment analyses performed for the cattle-yaks vs. 5-years-old yaks comparison revealed differentially methylated genes to be associated with GO terms related to protein ubiquitination, ubiquitin ligase complexes, ubiquitin-dependent protein catabolism, and endocytotic activity, as well as with KEGG terms related to apoptosis and the Fanconi anemia pathway. Overall, enrichment analyses for the cattle-yaks vs. 18-months-old yaks comparison were primarily associated with spermatogenesis, whereas those for the cattle-yaks vs. 5-years-old yaks comparison were primarily associated with apoptosis.

2 5’ Untranslated Region-Based Detection of Genetically Diverse APPV Strains in US Midwest

Abstract Atypical porcine pestivirus (APPV), an RNA virus member of the Flaviviridae family, has been associated with congenital tremor in newborn piglets. Previous qPCR-based assays, targeting the polyprotein coding sequences of APPV, were unable to detect the virus in novel cases of congenital tremor originated from multiple swine farms from the U.S. Midwest (MW). The polyprotein coding genes of APPV were characterized by a low level of sequence conservation ranging from 58.2 to 70.7% among 15 global APPV strains. In contrast, the 5’ untranslated region (UTR) was found to have a much greater degree of sequence conservation of 81.0% seen among the 15 global and new 4 MW strains, and as high as 91.5% when comparing only 5 MW strains. The complete 5’ UTR sequence of the APPV strains originated from MW was obtained by utilizing the template switching approach followed by amplification and dideoxy sequencing. Notably, previously identified potentially crucial regions for the correct function of the APPV internal ribosome entry site (IRES) were found to overlap with the most highly conserved 5’UTR segments, suggesting their functional role in viral protein translation. By targeting 100% conserved 5’UTR regions across 19 global and MW strains, a new qPCR assay was designed which was able to detect APPV in well documented cases of congenital tremor which originated from 5 MW farm sites (1-18 samples/site). The assay was found to have a detection sensitivity of 6.5 APPV copies (Cq ~38.4). Due to the potential functional importance of the highly conserved 5’UTR sequences targeted by our newly developed qPCR assay, we expect that assays targeting this region would broadly detect APPV strains that are diverse in space and time.

LncRNAs and Chromatin Modifications Pattern m6A Methylation at the Untranslated Regions of mRNAs.

New roles for RNA in mediating gene expression are being discovered at an alarming rate. A broad array of pathways control patterning of N6-methyladenosine (m6A) methylation on RNA transcripts. This review comprehensively discusses long non-coding RNAs (lncRNAs) as an additional dynamic regulator of m6A methylation, with a focus on the untranslated regions (UTRs) of mRNAs. Although there is extensive literature describing m6A modification of lncRNA, the function of lncRNA in guiding m6A writers has not been thoroughly explored. The independent control of lncRNA expression, its heterogeneous roles in RNA metabolism, and its interactions with epigenetic machinery, alludes to their potential in dynamic patterning of m6A methylation. While epigenetic regulation by histone modification of H3K36me3 has been demonstrated to pattern RNA m6A methylation, these modifications were specific to the coding and 3′UTR regions. However, there are observations that 5′UTR m6A is distinct from that of the coding and 3′UTR regions, and substantial evidence supports the active regulation of 5′UTR m6A methylation. Consequently, two potential mechanisms in patterning the UTRs m6A methylation are discussed; (1) Anti-sense lncRNA (AS-lncRNA) can either bind directly to the UTR, or (2) act indirectly via recruitment of chromatin-modifying complexes to pattern m6A. Both pathways can guide the m6A writer complex, facilitate m6A methylation and modulate protein translation. Findings in the lncRNA-histone-m6A axis could potentially contribute to the discovery of new functions of lncRNAs and clarify lncRNA-m6A findings in translational medicine.

MiR-424 inhibits apoptosis and inflammatory responses induced by sevoflurane through TLR4/MyD88/NF-\u03baB pathway

BackgroundSide effects of sevoflurane in anterograde and retrograde memory have been widely reported. However, there is no convincing evidence that sevoflurane directly causes the development of neurotoxicity. miR-424 has the potential to regulate the neurotoxicity caused by isoflurane anesthesia, and it has a complementary sequence with the 3’UTR region of TLR4. Thus, our study aims to explore whether sevoflurane directly causes neurotoxicity, the effects of miR-424 on sevoflurane induced apoptosis and inflammation, and the underlying mechanism.MethodsSevoflurane effects were identified both in mouse and in PC12 cells. Western blots and ELISA were used for protein detection, while micro (mi) RNA expression was measured with RT-qPCR. Dual luciferase reporter assays were employed to study the interaction between miR-424 and Toll-like receptor 4 (TLR4) using miR-424 mimics and TLR4 over-expression.ResultsSevoflurane stimulated expression of Bax2 and Caspase-3, and increased apoptosis ratio both in vivo and vitro (P < 0.05). Inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, were up-regulated by sevoflurane, while IL-10 was downregulated (P < 0.05). Sevoflurane treatment enhanced the phosphorylation of NF-κB, and up-regulated the expressions of TLR4 and MyD88 (P < 0.05), which demonstrated that sevoflurane inhibited proliferation and differentiation of neuronal cells by activating TLR4/MyD88/NF-κB signaling both in vitro and vivo. However, up-regulation of miR-424 attenuated the negative effects of sevoflurane by targeting the 3′-untranslated region (UTR) of TLR4 and inducing the degradation of mRNA (P < 0.05).ConclusionsIn vitro, sevoflurane induces activation of the endogenous TLR4 signaling pathway, thereby promoting apoptosis and inflammatory cytokine expression. Exogenous TLR4 acts as an agonist to stimulate TLR4 signaling, whereas miR-424 inhibits both endogenous and exogenous TLR4 signaling, thereby preserving proliferation and differentiation of neuronal cells.

MicroRNA‑15a promotes prostate cancer cell ferroptosis by inhibiting GPX4 expression

Ferroptosis is a novel form of regulated cell death characterized by accumulated lipid reactive oxygen species (ROS) and inactivation of glutathione peroxidase 4 (GPX4). The present study aimed to investigate the role of microRNA (miRNA/miR)-15a in ferroptosis of prostate cancer cells. Bioinformatics analysis was performed to predict the potential interaction between miR-15a and the 3'-untranslated region (UTR) of GPX4 mRNA. The prostate cancer cell line, LNCAP was transfected with miR-15a mimics or small interfering (si)-GPX4. Reverse transcription-quantitative PCR and western blot analyses were performed to detect the mRNA and protein expression levels of GPX4, respectively. Biotin-RNA pull-down and dual-luciferase reporter assays were performed to verify the interaction between miR-15a and GPX4 mRNA. The Cell Counting Kit-8 assay was performed to assess cell proliferation, while lactate dehydrogenase (LDH) and intracellular ferrous iron levels were detected via ELISA. Lipid ROS and mitochondrial membrane potential (MMP) were assessed via flow cytometry and staining with C11-BIODIPY probes or JC-1. Furthermore, lipid peroxidation was identified by measuring malondialdehyde (MDA) levels. The results demonstrated that transfection with miR-15a mimics decreased GPX4 protein expression. Bioinformatics analysis revealed potential binding sites between miR-15a and the 3'-UTR region of GPX4, and RNA pull-down and the dual-luciferase reporter assays further confirmed the interaction between miR-15a and GPX4 mRNA. Both transfection with miR-15a mimics and si-GPX4 suppressed cell proliferation, elevated LDH release, accumulated intracellular ferrous iron and ROS, disrupted MMP and increased MDA levels. Taken together, the results of the present study suggest miR-15a induces ferroptosis by regulating GPX4 in prostate cancer cells, which provides evidence for investigating the therapeutic strategies of prostate cancer.

Differential Expression of DDX3 and microRNAs in Response to Hormone and Cisplatin Against Cervical Cancer

Objectives: To discover micro ribonucleic acids (miRNAs) involved in the regulation of DDX3 expression using sexual hormones in combination with the well-known anticancer medication cisplatin. Methods: SiHa cells were treated with estradiol, dihydrotestosterone, and cisplatin and evaluated the expression of ER beta, Ki67, and DDX3 via quantitative reverse transcription–polymerase chain reaction. We generated a chimeric fusion construct five untranslated region (UTR)–hLUC–3UTR in the pEZX-MT06 miRNA vector under the control of the SV40 promoter. Reporter activity is measured with/without hormones, and their activity is compared with 5'- and 3'-UTR respectively. Various reporter deletion constructs were generated to identify the minimal UTR region in regulating the expression of DDX3. We identified the potential miRNA binding sites on the DDX3 UTR region, and their expression is monitored in cancer patients and cisplatin-treated SiHa cells. Results: Hormones increased the proliferation of SiHa cells and expression of DDX3. The 3'-UTR region 2135–4307bp contains miRNA sites that regulate DDX3 expression. miRNAs hsa-miR-671-5p, hsa-miR-361-5p, hsa-miR-140-5p, hsamiR-564, and hsa-miR-769-5p downregulated in patient samples but upregulated in cisplatin-treated cells. miRNA hsamiR-671-5p and hsa-miR-564 were associated with patient data and cisplatin-treated cancer cells. Conclusion: We discovered that sexual hormones enhanced DDX3 expression in SiHa cells. MiR-671-5p and miR-564 are two potential therapeutic miRNAs that can be used to treat DDX3-related malignancies. Keywords: Cancer patients, Cervical cancer, Cisplatin, DDX3, miRNA

Genetic variability in 5' untranslated region of BMPR1B gene from prolific Avishaan strain of sheep

Untranslated regions (UTRs) of the mRNAs have significant contribution towards gene regulation and expression. FecB gene is a member of transforming growth factor-p superfamily. A mutation in exon 8 of gene gives the FecB phenotype in sheep. In the present study 5′UTR region (992 bp encompassing 5′UTR and upstream sequences) and exon 8 region (712 bp encompassing exon 8 and partial sequences of upstream and downstream introns) of FecB gene were sequenced and characterized in prolific Avishaan sheep and its parental stock i.e. Garole, Malpura and Patanwadi. Sequence analysis of 992 bp fragment revealed 14 SNPs and one non-synonymous mutation (rs402422707) was observed in exon 1. Promoter was located in nucleotide 40 to 90 in amplified 5′ UTR sequence. Transcription factor binding site prediction identified 65 segments in the amplified 5′UTR and 5′ upstream sequence as potential binding sites for different transcription factors (TFs). In all 37 TFs binding sites were located on these sequences and majority of these sites were identified for SP-1 (12 segments), c/EBPalp (9 segments), NF-1 (4 segments), 2 segments each of RAP-1, HNF-1, c-Ets-1, TEC1, and Pit1a. These potential binding sites for TFs would be helpful in identifying site of interaction to control gene expression in Avishaan sheep.

Pax-5 Protein Expression Is Regulated by Transcriptional 3′UTR Editing

The Pax-5 gene encodes a transcription factor that is essential for B-cell commitment and maturation. However, Pax-5 deregulation is associated with various cancer lesions, notably hematopoietic cancers. Mechanistically, studies have characterized genetic alterations within the Pax-5 locus that result in either dominant oncogenic function or haploinsufficiency-inducing mutations leading to oncogenesis. Apart from these mutations, some examples of aberrant Pax-5 expression cannot be associated with genetic alterations. In the present study, we set out to elucidate potential alterations in post-transcriptional regulation of Pax-5 expression and establish that Pax-5 transcript editing represents an important means to aberrant expression. Upon the profiling of Pax-5 mRNA in leukemic cells, we found that the 3′end of the Pax-5 transcript is submitted to alternative polyadenylation (APA) and alternative splicing events. Using rapid amplification of cDNA ends (3′RACE) from polysomal fractions, we found that Pax-5 3′ untranslated region (UTR) shortening correlates with increased ribosomal occupancy for translation. These observations were also validated using reporter gene assays with truncated 3′UTR regions cloned downstream of a luciferase gene. We also showed that Pax-5 3′UTR editing has direct repercussions on regulatory elements such as miRNAs, which in turn impact Pax-5 protein expression. More importantly, we found that advanced staging of various hematopoietic cancer lesions relates to shorter Pax-5 3′UTRs. Altogether, our findings identify novel molecular mechanisms that account for aberrant expression and function of the Pax-5 oncogene in cancer cells. These findings also present new avenues for strategic intervention in Pax-5-mediated cancers.

High quality genome assembly of the amitochondriate eukaryote Monocercomonoides exilis.

Monocercomonoides exilis is considered the first known eukaryote to completely lack mitochondria. This conclusion is based primarily on a genomic and transcriptomic study which failed to identify any mitochondrial hallmark proteins. However, the available genome assembly has limited contiguity and around 1.5 % of the genome sequence is represented by unknown bases. To improve the contiguity, we re-sequenced the genome and transcriptome of M. exilis using Oxford Nanopore Technology (ONT). The resulting draft genome is assembled in 101 contigs with an N50 value of 1.38 Mbp, almost 20 times higher than the previously published assembly. Using a newly generated ONT transcriptome, we further improve the gene prediction and add high quality untranslated region (UTR) annotations, in which we identify two putative polyadenylation signals present in the 3′UTR regions and characterise the Kozak sequence in the 5′UTR regions. All these improvements are reflected by higher BUSCO genome completeness values. Regardless of an overall more complete genome assembly without missing bases and a better gene prediction, we still failed to identify any mitochondrial hallmark genes, thus further supporting the hypothesis on the absence of mitochondrion.

SNCA 3' UTR Genetic Variants in Patients with Parkinson's Disease.

The SNCA (Synuclein Alpha) gene represents a major risk gene for Parkinson’s disease (PD) and SNCA polymorphisms have been associated with the common sporadic form of PD. Numerous Genome-Wide Association Studies showed strong signals located in the SNCA 3′ UTR (untranslated region) region indicating that variants in 3′ UTRs of PD-associated genes could contribute to neurodegeneration and may regulate the risk for PD. Genetic variants in 3′ UTR can affect miRNA activity and consequently change the translation process. The aim of this study was to access the differences in 3′ UTR variants of SNCA genes in a cohort of PD patients and control subjects from Croatia. The cohort consisted of 52 PD patients and 23 healthy control subjects. Differences between 3′ UTR allele and genotype frequencies were accessed through next generation sequencing approach from whole blood samples. In our study, we identified four previously reported single nucleotide polymorphisms (SNPs) and one insertion in the 3′ UTR region of SNCA gene, namely rs1045722, rs3857053, rs577490090, rs356165, and rs777296100, and five variants not reported in the literature, namely rs35270750, rs529553259, rs377356638, rs571454522, and rs750347645. Our results indicate a significantly higher occurrence of the rs571454522 variant in the PD population. To the best of our knowledge, this variant has not been reported until now in the literature. We analyzed our results in the context of previous research, creating a brief overview of the importance of 3′ UTR variants of the SNCA gene. Further studies will be needed to gain a more profound insight regarding their role in PD development, which will help to assess the role and impact of post-transcriptional regulation on disease pathology.

The Influence of ICAM1 3'UTR Gene Polymorphism on the Occurrence and Metastasis of Primary Liver Cancer.

Objective In this study, we explored the influence of single nucleotide polymorphism (SNP) in the noncoding region of intercellular adhesion molecule 1 (ICAM1) gene on the occurrence and metastasis of primary hepatocellular carcinoma (PHC). Methods Sanger sequencing was used to analyze the genotypes of rs3093032, rs923366, and rs281437 locus in the 3′untranslated region (UTR) of the ICAM1 gene. The level of plasma ICAM1 was analyzed by enzyme-linked immunosorbent assay (ELISA). Results After adjusting for risk factors such as BMI, smoking, drinking, family history of tumors, and hepatitis B virus test results, the CT genotype at rs3093032 of the ICAM1 gene (OR = 0.19, 95% CI: 0.08-0.44, P < 0.01), dominance model (OR = 0.23, 95% CI: 0.11-0.48, P < 0.01), and T allele (OR = 0.27, 95% CI: 0.14-0.53, P < 0.01) were related to the reduced risk of PHC susceptibility. rs923366 locus CT genotype (OR = 0.63, 95% CI: 0.44-0.90, P = 0.01), TT genotype (OR = 0.23, 95% CI: 0.10-0.53, P < 0.01), dominant model (OR = 0.55, 95% CI: 0.39-0.77, P < 0.01), recessive model (OR = 0.28, 95% CI: 0.12-0.62, P < 0.01), and T allele (OR = 0.55, 95% CI: 0.42-0.73, P < 0.01) were related to a reduction in the risk of PHC susceptibility. rs281437 locus CT genotype (OR = 2.08, 95% CI: 1.40-3.09, P < 0.01), TT genotype (OR = 5.20, 95% CI: 2.22-12.17, P < 0.01), dominant model (OR = 2.45, 95% CI: 1.69-3.54, P < 0.01), recessive model (OR = 4.32, 95% CI: 1.86-10.06, P < 0.01), and T allele (OR = 2.46, 95% CI: 1.79-3.38, P < 0.01) were significantly related to the increased risk of PHC susceptibility. SNPs at rs3093032, rs923366, and rs281437 of the ICAM1 gene were significantly correlated with TNM stage and tumor metastasis of PHC patients (P < 0.05). Conclusion SNPs at rs3093032, rs923366, and rs281437 in the 3′UTR region of the ICAM1 gene are related to the occurrence and metastasis of PHC.