Analysis of Alternative LDLRAD4 Gene Promoters and Transcripts in Colorectal Cancer

Gene LDLRAD4 plays a role in cell proliferation, apoptosis, immunosuppression and cancer progression. Transcription of LDLRAD4 is regulated by several alternative promoters, two of which were indicated by in silico analyses to be differentially active in rectal cancer. Promoter A encodes for a truncated protein-coding transcript and is down-regulated in rectal cancer. Promoter B encodes for a non-coding transcript up-regulated in rectal cancer identified as lnc-RNMT-2:5. The aim of this study was to characterize the two alternative promoters in silico in order to explain their differential activity and to investigate the profile of LDLRAD4 transcripts in colon cell lines. Nucleotide sequences used in the analyses were downloaded from the Ensemble genome database (reference GRCh37). Three bioinformatics tools were used for core promoter element prediction: GPMiner, YAPP and CNNPromoter. Four bioinformatics tools were used for transcription factor binding site prediction: PROMO, TFBIND, CiiiDER and Tfsitescan. Only the predictions made by two or more tools were considered. Primer extension followed by fragment analysis was used to characterize LDLRAD4 transcripts present in colon cell lines. The promoter element predictions showed that the promoter A is typical, while promoter B has most typical elements and lacks GC boxes. The transcription binding site predictions indicate that three different transcription factors bind only to the promoter A (NF-kB, EGR1 and IRF-7), while four different transcription factors bind only to the promoter B (HNF1, POU2F1, POU2F2 and PTF1). The predicted transcription factors are mostly involved in regulation of cell differentiation and proliferation. The primer extension experiment performed with primer specific for exon 2-exon 3 junction produced multiple signals of relatively low intensity, indicating the presence of multiple LDLRAD4 transcripts in colon cell lines. The results obtained by in silico analysis may explain promoter B activation in rectal cancer. However, based on the results of primer extension, neither of the LDLRAD4 transcripts is dominant in colon cell lines. Considering that promoter B generates long non-coding RNA that can exert its function even at low expression level, it can serve as potential colorectal cancer biomarker and its potential role in carcinogenesis should be investigated

Long read sequencing – the next level in genomic research

Sekvenciranje dugih fragmenata ili treća generacija sekvenciranja u realnom vremenu produkuje očitavanja pojedinačnih molekula DNK dužine od 1 kb do nekoliko Mb sa očuvanim epigenetičkim oznakama. Dostupne tehnologije su sekvenciranje pojedinačnih molekula u realnom vremenu (eng. single-molecule real-time sequencing, PacBio) i sekvenciranje kroz proteinske nanopore (Oxford Nanopore Technologies). PacBio tehnologija zasnovana je na detekciji ugradnje nukelotida od strane pojedinačnog molekula DNK polimeraze u realnom vremenu, korišćenjem fluoresecencije kao surogat markera. PacBio HiFi očitavanja su dužine ~15 kb sa tačnošću >99,9%. Oxford Nanopore tehnologija izvodi sekvencu nukleotida iz promena u intenzitetu jonske struje dok DNK prolazi kroz stohastički senzor – proteinsku nanoporu.Može sekvencirati fragmente DNK u rasponu od pet redova veličina (20 bp do nekoliko Mb) sa tačnošću dupleks očitavanja >99,9% kada se koriste R10.4.1 nanopore. Sa elektronskim „čitanjem” nukleinskih kiselina, inovacije kao što su minijaturni uređaj veličine dlana sa cenom <1000 dolara, sekvenciranje na terenu, digitalno obogaćivanje ciljnih sekvenci (adaptivno uzorkovanje) i direktno sekvenciranje RNK, postali su stvarnost. Sekvenciranje dugih fragmenata omogućilo je kompletiranje sekvence genoma čoveka, objavljivanje drafta ljudskog pangenoma i ubrzalo je sekvenciranje genoma eukariota. Od uvođenja metode 2011. godine sekvencirano je ~1000 od 1065 genoma deponovanih u NCBI bazi. Puni potencijal metode u izučavanju transkriptoma i epigenoma biće vidljiv u godinama koje slede. Sekvenciranje dugih fragmenata postaje osnova precizne medicine efikasne za sve ljudske populacije i očuvanja biodiverziteta, i zavredelo je da bude metoda 2022. godine prema časopisu Nature Methods.Long read or third-generation sequencing produces reads from 1 kb to several Mb in length with preserved epigenetic marks, at the single-molecule level and in real-time. Single-molecule real-time sequencing (PacBio) and protein nanopore sequencing (Oxford Nanopore Technologies) are available technologies. PacBio technology is based on monitoring the nucleotide incorporation by a single DNA polymerase molecule in real time using fluorescence as a surrogate marker. PacBio HiFi reads are ~15 kb in length with >99.9% accuracy. Oxford Nanopore sequencing infers nucleotide sequence from the changes in ion current intensity while DNA passes through a stochastic sensor – a protein nanopore. It can sequence DNA fragments ranging in five orders of magnitude (20 bp to several Mb), with duplex read accuracy >99.9% when using R10.4.1 nanopores. Innovations such as a miniature device of the palm-size with a price <1000 dollars, sequencing in the field, digital enrichment of target sequences (adaptive sampling) and direct RNA sequencing have become a reality with the electronic „reading“ of nucleic acids. Long read sequencing enabled completing the human genome sequence and releasing a draft of the human pangenome reference. It has also accelerated genome sequencing of eukaryotic species. Out of 1065 genomes deposited in the NCBI database, ~1000 were sequenced since its development. The full potential of the method in studying transcriptome and epigenome will be visible in the years to come. Long read sequencing is becoming the basis of precision medicine effective for all human populations and biodiversity conservation and was announced as the method of the year 2022 according to Nature Methods