Alignment Of RNA Sequences Research Articles

RNA language models predict mutations that improve RNA function.

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. While advances in deep learning enable the prediction of accurate protein structural models, RNA structure prediction is not possible at present due to a lack of abundant high-quality reference data 1 . Furthermore, available sequence data are generally not associated with organismal phenotypes that could inform RNA function 2-4 . We created GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB) 5 . GARNET links RNA sequences derived from GTDB genomes to experimental and predicted optimal growth temperatures of GTDB reference organisms. This enables construction of deep and diverse RNA sequence alignments to be used for machine learning. Using GARNET, we define the minimal requirements for a sequence- and structure-aware RNA generative model. We also develop a GPT-like language model for RNA in which overlapping triplet tokenization provides optimal encoding. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identified mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB- derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

Higher Maternal Body Mass Index Is Associated With Lower Placental Expression of EPYC: A Genome-Wide Transcriptomic Study.

Elevated body mass index (BMI) in pregnancy is associated with adverse maternal and fetal outcomes. The placental transcriptome may elucidate molecular mechanisms underlying these associations. We examined the association of first-trimester maternal BMI with the placental transcriptome in the Gen3G prospective cohort. We enrolled participants at 5 to 16 weeks of gestation and measured height and weight. We collected placenta samples at delivery. We performed whole-genome RNA sequencing using Illumina HiSeq 4000 and aligned RNA sequences based on the GTEx v8 pipeline. We conducted differential gene expression analysis of over 15 000 genes from 450 placental samples and reported the change in normalized gene expression per 1-unit increase in log2 BMI (kg/m2) as a continuous variable using Limma Voom. We adjusted models for maternal age, fetal sex, gestational age at delivery, gravidity, and surrogate variables accounting for technical variability. We compared participants with BMI of 18.5 to 24.9 mg/kg2 (N = 257) vs those with obesity (BMI ≥30 kg/m2, N = 82) in secondary analyses. Participants' mean ± SD age was 28.2 ± 4.4 years and BMI was 25.4 ± 5.5 kg/m2 in early pregnancy. Higher maternal BMI was associated with lower placental expression of EPYC (slope = -1.94, false discovery rate [FDR]-adjusted P = 7.3 × 10-6 for continuous BMI; log2 fold change = -1.35, FDR-adjusted P = 3.4 × 10-3 for BMI ≥30 vs BMI 18.5-24.9 kg/m2) and with higher placental expression of IGFBP6, CHRDL1, and CXCL13 after adjustment for covariates and accounting for multiple testing (FDR < 0.05). Our genome-wide transcriptomic study revealed novel genes potentially implicated in placental biologic response to higher maternal BMI in early pregnancy.

GINGER: an integrated method for high-accuracy prediction of gene structure in higher eukaryotes at the gene and exon level.

The prediction of gene structure within the genome sequence is the starting point of genome analysis, and its accuracy has a significant impact on the quality of subsequent analyses. Gene structure prediction is roughly divided into RNA-Seq-based methods, ab initio-based methods, homology-based methods, and the integration of individual prediction methods. Integrated methods are mainstream in recent genome projects because they improve prediction accuracy by combining or taking the best individual prediction findings; however, adequate prediction accuracy for eukaryotic species has not yet been achieved. Therefore, we developed an integrated tool, GINGER, that solves various issues related to gene structure prediction in higher eukaryotes. By handling artefacts in alignments of RNA and protein sequences, reconstructing gene structures via dynamic programming with appropriately weighted and scored exon/intron/intergenic regions, and applying different prediction processes and filtering criteria to multi-exon and single-exon genes, we achieved a significant improvement in accuracy compared to the existing integration methods. The feature of GINGER is its high prediction accuracy at the gene and exon levels, which is pronounced for species with more complex gene architectures. GINGER is implemented using Nextflow, which allows for the efficient and effective use of computing resources.

RNA covariation at helix-level resolution for the identification of evolutionarily conserved RNA structure.

Many biologically important RNAs fold into specific 3D structures conserved through evolution. Knowing when an RNA sequence includes a conserved RNA structure that could lead to new biology is not trivial and depends on clues left behind by conservation in the form of covariation and variation. For that purpose, the R-scape statistical test was created to identify from alignments of RNA sequences, the base pairs that significantly covary above phylogenetic expectation. R-scape treats base pairs as independent units. However, RNA base pairs do not occur in isolation. The Watson-Crick (WC) base pairs stack together forming helices that constitute the scaffold that facilitates the formation of the non-WC base pairs, and ultimately the complete 3D structure. The helix-forming WC base pairs carry most of the covariation signal in an RNA structure. Here, I introduce a new measure of statistically significant covariation at helix-level by aggregation of the covariation significance and covariation power calculated at base-pair-level resolution. Performance benchmarks show that helix-level aggregated covariation increases sensitivity in the detection of evolutionarily conserved RNA structure without sacrificing specificity. This additional helix-level sensitivity reveals an artifact that results from using covariation to build an alignment for a hypothetical structure and then testing the alignment for whether its covariation significantly supports the structure. Helix-level reanalysis of the evolutionary evidence for a selection of long non-coding RNAs (lncRNAs) reinforces the evidence against these lncRNAs having a conserved secondary structure.

Network-Based Structural Alignment of RNA Sequences Using TOPAS.

TOPAS (TOPological network-based Alignment of Structural RNAs) is a network-based alignment algorithm that predicts structurally sound pairwise alignment of RNAs. In order to take advantage of recent advances in comparative network analysis for efficient structurally sound RNA alignment, TOPAS constructs topological network representations for RNAs, which consist of sequential edges connecting nucleotide bases as well as structural edges reflecting the underlying folding structure. Structural edges are weighted by the estimated base-pairing probabilities. Next, the constructed networks are aligned using probabilistic network alignment techniques, which yield a structurally sound RNA alignment that considers both the sequence similarity and the structural similarity between the given RNAs. Compared to traditional Sankoff-style algorithms, this network-based alignment scheme leads to a significant reduction in the overall computational cost while yielding favorable alignment results. Another important benefit is its capability to handle arbitrary folding structures, which can potentially lead to more accurate alignment for RNAs with pseudoknots.

A curated human cellular microRNAome based on 196 primary cell types.

BackgroundAn incomplete picture of the expression distribution of microRNAs (miRNAs) across human cell types has long hindered our understanding of this important regulatory class of RNA. With the continued increase in available public small RNA sequencing datasets, there is an opportunity to more fully understand the general distribution of miRNAs at the cell level.ResultsFrom the NCBI Sequence Read Archive, we obtained 6,054 human primary cell datasets and processed 4,184 of them through the miRge3.0 small RNA sequencing alignment software. This dataset was curated down, through shared miRNA expression patterns, to 2,077 samples from 196 unique cell types derived from 175 separate studies. Of 2,731 putative miRNAs listed in miRBase (v22.1), 2,452 (89.8%) were detected. Among reasonably expressed miRNAs, 108 were designated as cell specific/near specific, 59 as infrequent, 52 as frequent, 54 as near ubiquitous, and 50 as ubiquitous. The complexity of cellular microRNA expression estimates recapitulates tissue expression patterns and informs on the miRNA composition of plasma.ConclusionsThis study represents the most complete reference, to date, of miRNA expression patterns by primary cell type. The data are available through the human cellular microRNAome track at the UCSC Genome Browser (https://genome.ucsc.edu/cgi-bin/hgHubConnect) and an R/Bioconductor package (https://bioconductor.org/packages/microRNAome/).

KnotAli: informed energy minimization through the use of evolutionary information

BackgroundImproving the prediction of structures, especially those containing pseudoknots (structures with crossing base pairs) is an ongoing challenge. Homology-based methods utilize structural similarities within a family to predict the structure. However, their prediction is limited to the consensus structure, and by the quality of the alignment. Minimum free energy (MFE) based methods, on the other hand, do not rely on familial information and can predict structures of novel RNA molecules. Their prediction normally suffers from inaccuracies due to their underlying energy parameters.ResultsWe present a new method for prediction of RNA pseudoknotted secondary structures that combines the strengths of MFE prediction and alignment-based methods. KnotAli takes a multiple RNA sequence alignment as input and uses covariation and thermodynamic energy minimization to predict possibly pseudoknotted secondary structures for each individual sequence in the alignment. We compared KnotAli’s performance to that of three other alignment-based programs, two that can handle pseudoknotted structures and one control, on a large data set of 3034 RNA sequences with varying lengths and levels of sequence conservation from 10 families with pseudoknotted and pseudoknot-free reference structures. We produced sequence alignments for each family using two well-known sequence aligners (MUSCLE and MAFFT).ConclusionsWe found KnotAli’s performance to be superior in 6 of the 10 families for MUSCLE and 7 of the 10 for MAFFT. While both KnotAli and Cacofold use background noise correction strategies, we found KnotAli’s predictions to be less dependent on the alignment quality. KnotAli can be found online at the Zenodo image: https://doi.org/10.5281/zenodo.5794719

Informative RNA base embedding for RNA structural alignment and clustering by deep representation learning.

Effective embedding is actively conducted by applying deep learning to biomolecular information. Obtaining better embeddings enhances the quality of downstream analyses, such as DNA sequence motif detection and protein function prediction. In this study, we adopt a pre-training algorithm for the effective embedding of RNA bases to acquire semantically rich representations and apply this algorithm to two fundamental RNA sequence problems: structural alignment and clustering. By using the pre-training algorithm to embed the four bases of RNA in a position-dependent manner using a large number of RNA sequences from various RNA families, a context-sensitive embedding representation is obtained. As a result, not only base information but also secondary structure and context information of RNA sequences are embedded for each base. We call this ‘informative base embedding’ and use it to achieve accuracies superior to those of existing state-of-the-art methods on RNA structural alignment and RNA family clustering tasks. Furthermore, upon performing RNA sequence alignment by combining this informative base embedding with a simple Needleman–Wunsch alignment algorithm, we succeed in calculating structural alignments with a time complexity of O(n2) instead of the O(n6) time complexity of the naive implementation of Sankoff-style algorithm for input RNA sequence of length n.

LaRA 2: parallel and vectorized program for sequence\u2013structure alignment of RNA sequences

BackgroundThe function of non-coding RNA sequences is largely determined by their spatial conformation, namely the secondary structure of the molecule, formed by Watson–Crick interactions between nucleotides. Hence, modern RNA alignment algorithms routinely take structural information into account. In order to discover yet unknown RNA families and infer their possible functions, the structural alignment of RNAs is an essential task. This task demands a lot of computational resources, especially for aligning many long sequences, and it therefore requires efficient algorithms that utilize modern hardware when available. A subset of the secondary structures contains overlapping interactions (called pseudoknots), which add additional complexity to the problem and are often ignored in available software.ResultsWe present the SeqAn-based software LaRA 2 that is significantly faster than comparable software for accurate pairwise and multiple alignments of structured RNA sequences. In contrast to other programs our approach can handle arbitrary pseudoknots. As an improved re-implementation of the LaRA tool for structural alignments, LaRA 2 uses multi-threading and vectorization for parallel execution and a new heuristic for computing a lower boundary of the solution. Our algorithmic improvements yield a program that is up to 130 times faster than the previous version.ConclusionsWith LaRA 2 we provide a tool to analyse large sets of RNA secondary structures in relatively short time, based on structural alignment. The produced alignments can be used to derive structural motifs for the search in genomic databases.

ConsAlifold: considering RNA structural alignments improves prediction accuracy of RNA consensus secondary structures.

By detecting homology among RNAs, the probabilistic consideration of RNA structural alignments has improved the prediction accuracy of significant RNA prediction problems. Predicting an RNA consensus secondary structure from an RNA sequence alignment is a fundamental research objective because in the detection of conserved base-pairings among RNA homologs, predicting an RNA consensus secondary structure is more convenient than predicting an RNA structural alignment. We developed and implemented ConsAlifold, a dynamic programming-based method that predicts the consensus secondary structure of an RNA sequence alignment. ConsAlifold considers RNA structural alignments. ConsAlifold achieves moderate running time and the best prediction accuracy of RNA consensus secondary structures among available prediction methods. ConsAlifold, data and Python scripts for generating both figures and tables are freely available at https://github.com/heartsh/consalifold. Supplementary data are available at Bioinformatics online.

Development of fluorescence in situ hybridization (FISH) probes to detect and enumerate Gambierdiscus species

Ciguatera poisoning (CP) is a syndrome caused by the bioaccumulation of lipophilic ciguatoxins in coral reef fish and invertebrates, and their subsequent consumption by humans. These phycotoxins are produced by Gambierdiscus spp., tropical epiphytic dinoflagellates that live on a variety of macrophytes, as well as on dead corals and sand. Recent taxonomic studies have identified novel diversity within the Gambierdiscus genus, with at least 18 species and several sub-groups now identified, many of which co-occur and differ significantly in toxicity. The ability to accurately and quickly distinguish Gambierdiscus species in field samples and determine community composition and abundance is central to assessing CP risk, yet most Gambierdiscus species are indistinguishable using light microscopy, and other enumeration methods are semi-quantitative. In order to investigate the spatial and temporal dynamics of Gambierdiscus species and community toxicity, new tools for species identification and enumeration in field samples are needed. Here, fluorescence in situ hybridization (FISH) probes were designed for seven species commonly found in the Caribbean Sea and Pacific Ocean, permitting their enumeration in field samples using epifluorescence microscopy. This technique enables the assessment of community composition and accurate determination of cell abundances of individual species. Molecular probes detecting G. australes, G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, and the G. silvae/G. polynesiensis clade were designed using alignments of large subunit ribosomal RNA (rRNA) sequences. These probes were tested for specificity and cross-reactivity through experiments in which field samples were spiked with known concentrations of Gambierdiscus cultures, and analyzed to confirm that Gambierdiscus can be successfully detected and enumerated by FISH in the presence of detritus and other organisms. These probes were then used to characterize Gambierdiscus community structure in field samples collected from the Florida Keys and Hawai‘i, USA. The probes revealed the co-occurrence of multiple species at each location. Time-series FISH analyses of samples collected from the Florida Keys quantified seasonal shifts in community composition as well as fluctuations in overall Gambierdiscus cell abundance. Application of species-specific FISH probes provides a powerful new tool to those seeking to target individual Gambierdiscus species, including significant toxin-producers, in field populations. Moving forward, analysis of Gambierdiscus community composition across multiple environments and over time will also allow species dynamics to be linked to environmental parameters, improving our ability to understand and manage the current and changing risks of CP worldwide.

Deep forest ensemble learning for classification of alignments of non-coding RNA sequences based on multi-view structure representations.

Non-coding RNAs (ncRNAs) play crucial roles in multiple biological processes. However, only a few ncRNAs’ functions have been well studied. Given the significance of ncRNAs classification for understanding ncRNAs’ functions, more and more computational methods have been introduced to improve the classification automatically and accurately. In this paper, based on a convolutional neural network and a deep forest algorithm, multi-grained cascade forest (GcForest), we propose a novel deep fusion learning framework, GcForest fusion method (GCFM), to classify alignments of ncRNA sequences for accurate clustering of ncRNAs. GCFM integrates a multi-view structure feature representation including sequence-structure alignment encoding, structure image representation and shape alignment encoding of structural subunits, enabling us to capture the potential specificity between ncRNAs. For the classification of pairwise alignment of two ncRNA sequences, the F-value of GCFM improves 6% than an existing alignment-based method. Furthermore, the clustering of ncRNA families is carried out based on the classification matrix generated from GCFM. Results suggest better performance (with 20% accuracy improved) than existing ncRNA clustering methods (RNAclust, Ensembleclust and CNNclust). Additionally, we apply GCFM to construct a phylogenetic tree of ncRNA and predict the probability of interactions between RNAs. Most ncRNAs are located correctly in the phylogenetic tree, and the prediction accuracy of RNA interaction is 90.63%. A web server (http://bmbl.sdstate.edu/gcfm/) is developed to maximize its availability, and the source code and related data are available at the same URL.

Aligning biological sequences by exploiting residue conservation and coevolution.

Sequences of nucleotides (for DNA and RNA) or amino acids (for proteins) are central objects in biology. Among the most important computational problems is that of sequence alignment, i.e., arranging sequences from different organisms in such a way to identify similar regions, to detect evolutionary relationships between sequences, and to predict biomolecular structure and function. This is typically addressed through profile models, which capture position specificities like conservation in sequences but assume an independent evolution of different positions. Over recent years, it has been well established that coevolution of different amino-acid positions is essential for maintaining three-dimensional structure and function. Modeling approaches based on inverse statistical physics can catch the coevolution signal in sequence ensembles, and they are now widely used in predicting protein structure, protein-protein interactions, and mutational landscapes. Here, we present DCAlign, an efficient alignment algorithm based on an approximate message-passing strategy, which is able to overcome the limitations of profile models, to include coevolution among positions in a general way, and to be therefore universally applicable to protein- and RNA-sequence alignment without the need of using complementary structural information. The potential of DCAlign is carefully explored using well-controlled simulated data, as well as real protein and RNA sequences.

Insertions of codons encoding basic amino acids in H7 hemagglutinins of influenza A viruses occur by recombination with RNA at hotspots near snoRNA binding sites.

The presence of multiple basic amino acids in the protease cleavage site of the hemagglutinin (HA) protein is the main molecular determinant of virulence of highly pathogenic avian influenza (HPAI) viruses. Recombination of HA RNA with other RNA molecules of host or virus origin is a dominant mechanism of multibasic cleavage site (MBCS) acquisition for H7 subtype HA. Using alignments of HA RNA sequences from documented cases of MBCS insertion due to recombination, we show that such recombination with host RNAs is most likely to occur at particular hotspots in ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and viral RNAs. The locations of these hotspots in highly abundant RNAs indicate that RNA recombination is facilitated by the binding of small nucleolar RNA (snoRNA) near the recombination points.

Identification of important miRNAs in bubaline mammary gland at heifer stage - An in-silico approach

The present study was undertaken to identify the microRNAs (miRNAs) expressed in the mammary tissue of a bubaline heifer. Small RNAs were isolated from the mammary gland tissue and enriched for miRNA fraction. The linker-ligated small RNAs were reverse transcribed to synthesize cDNA and amplified by PCR. The PCR products were ligated to the pGEM-T Easy vector; cloned into DH5 alpha cells and sequenced. Sequencing of 40 clones, randomly chosen from this library, produced 115 concatemerized short sequences. The short sequences were checked for their matches with the help of refseq_rna database, EST database (NCBI) and Ensembl. The analysis was performed for more than 90% identity with miRNAs across different species in miRBase. Alignment of putative small RNA sequences with the bovine genome was attempted in GenBank (NCBI) and Ensembl using BLAST. The small RNA sequences with a partial matches within the bovine genome and/or with flanking sequences (upstream or downstream) were analyzed for hairpin structures using the Mfold web server. Reverse complements were also assessed for the homology search. miRNA sequences showing only a partial match with already reported sequences were considered as a putative bubaline miRNAs. Six developmentally important putative miRNA precursors were identified from this study using cloning and sequencing followed by the Bioinformatics approach. This study will help in the elucidation of pathways involving miRNAs in bubaline species at the heifer stage.

Evaluation of Seven Different RNA-Seq Alignment Tools Based on Experimental Data from the Model Plant Arabidopsis thaliana.

Quantification of gene expression is crucial to connect genome sequences with phenotypic and physiological data. RNA-Sequencing (RNA-Seq) has taken a prominent role in the study of transcriptomic reactions of plants to various environmental and genetic perturbations. However, comparative tests of different tools for RNA-Seq read mapping and quantification have been mainly performed on data from animals or humans, which necessarily neglect, for example, the large genetic variability among natural accessions within plant species. Here, we compared seven computational tools for their ability to map and quantify Illumina single-end reads from the Arabidopsis thaliana accessions Columbia-0 (Col-0) and N14. Between 92.4% and 99.5% of all reads were mapped to the reference genome or transcriptome and the raw count distributions obtained from the different mappers were highly correlated. Using the software DESeq2 to determine differential gene expression (DGE) between plants exposed to 20 °C or 4 °C from these read counts showed a large pairwise overlap between the mappers. Interestingly, when the commercial CLC software was used with its own DGE module instead of DESeq2, strongly diverging results were obtained. All tested mappers provided highly similar results for mapping Illumina reads of two polymorphic Arabidopsis accessions to the reference genome or transcriptome and for the determination of DGE when the same software was used for processing.

Structural Alignment and Covariation Analysis of RNA Sequences.

RNA molecules adopt defined structural conformations that are essential to exert their function. During the course of evolution, the structure of a given RNA can be maintained via compensatory base-pair changes that occur among covarying nucleotides in paired regions. Therefore, for comparative, structural, and evolutionary studies of RNA molecules, numerous computational tools have been developed to incorporate structural information into sequence alignments and a number of tools have been developed to study covariation. The bioinformatic protocol presented here explains how to use some of these tools to generate a secondary-structure-aware multiple alignment of RNA sequences and to annotate the alignment to examine the conservation and covariation of structural elements among the sequences.

RNA secondary structure prediction using an ensemble of two-dimensional deep neural networks and transfer learning

The majority of our human genome transcribes into noncoding RNAs with unknown structures and functions. Obtaining functional clues for noncoding RNAs requires accurate base-pairing or secondary-structure prediction. However, the performance of such predictions by current folding-based algorithms has been stagnated for more than a decade. Here, we propose the use of deep contextual learning for base-pair prediction including those noncanonical and non-nested (pseudoknot) base pairs stabilized by tertiary interactions. Since only <250 nonredundant, high-resolution RNA structures are available for model training, we utilize transfer learning from a model initially trained with a recent high-quality bpRNA dataset of > 10,000 nonredundant RNAs made available through comparative analysis. The resulting method achieves large, statistically significant improvement in predicting all base pairs, noncanonical and non-nested base pairs in particular. The proposed method (SPOT-RNA), with a freely available server and standalone software, should be useful for improving RNA structure modeling, sequence alignment, and functional annotations.

Evolutionary Analyses of Base-Pairing Interactions in DNA and RNA Secondary Structures.

Pairs of nucleotides within functional nucleic acid secondary structures often display evidence of coevolution that is consistent with the maintenance of base-pairing. Here, we introduce a sequence evolution model, MESSI (Modeling the Evolution of Secondary Structure Interactions), that infers coevolution associated with base-paired sites in DNA or RNA sequence alignments. MESSI can estimate coevolution while accounting for an unknown secondary structure. MESSI can also use graphics processing unit parallelism to increase computational speed. We used MESSI to infer coevolution associated with GC, AU (AT in DNA), GU (GT in DNA) pairs in noncoding RNA alignments, and in single-stranded RNA and DNA virus alignments. Estimates of GU pair coevolution were found to be higher at base-paired sites in single-stranded RNA viruses and noncoding RNAs than estimates of GT pair coevolution in single-stranded DNA viruses. A potential biophysical explanation is that GT pairs do not stabilize DNA secondary structures to the same extent that GU pairs do in RNA. Additionally, MESSI estimates the degrees of coevolution at individual base-paired sites in an alignment. These estimates were computed for a SHAPE-MaP-determined HIV-1 NL4-3 RNA secondary structure. We found that estimates of coevolution were more strongly correlated with experimentally determined SHAPE-MaP pairing scores than three nonevolutionary measures of base-pairing covariation. To assist researchers in prioritizing substructures with potential functionality, MESSI automatically ranks substructures by degrees of coevolution at base-paired sites within them. Such a ranking was created for an HIV-1 subtype B alignment, revealing an excess of top-ranking substructures that have been previously identified as having structure-related functional importance, among several uncharacterized top-ranking substructures.

A functional role for the cancer disparity-linked genes, CRY\u03b2B2 and CRY\u03b2B2P1, in the promotion of breast cancer

BackgroundIn the USA, the breast cancer mortality rate is 41% higher for African-American women than non-Hispanic White women. While numerous gene expression studies have classified biological features that vary by race and may contribute to poorer outcomes, few studies have experimentally tested these associations. CRYβB2 gene expression has drawn particular interest because of its association with overall survival and African-American ethnicity in multiple cancers. Several reports indicate that overexpression of the CRYβB2 pseudogene, CRYβB2P1, and not CRYβB2 is linked with race and poor outcome. It remains unclear whether either or both genes are linked to breast cancer outcomes. This study investigates CRYβB2 and CRYβB2P1 expression in human breast cancers and breast cancer cell line models, with the goal of elucidating the mechanistic contribution of CRYβB2 and CRYβB2P1 to racial disparities.MethodsCustom scripts for CRYβB2 or CRYβB2P1 were generated and used to identify reads that uniquely aligned to either gene. Gene expression according to race and tumor subtype were assessed using all available TCGA breast cancer RNA sequencing alignment samples (n = 1221). In addition, triple-negative breast cancer models engineered to have each gene overexpressed or knocked out were developed and evaluated by in vitro, biochemical, and in vivo assays to identify biological functions.ResultsWe provide evidence that CRYβB2P1 is expressed at higher levels in breast tumors compared to CRYβB2, but only CRYβB2P1 is significantly increased in African-American tumors relative to White American tumors. We show that independent of CRYβB2, CRYβB2P1 enhances tumorigenesis in vivo via promoting cell proliferation. Our data also reveal that CRYβB2P1 may function as a non-coding RNA to regulate CRYβB2 expression. A key observation is that the combined overexpression of both genes was found to suppress cell growth. CRYβB2 overexpression in triple-negative breast cancers increases invasive cellular behaviors, tumor growth, IL6 production, immune cell chemoattraction, and the expression of metastasis-associated genes. These data underscore that both CRYβB2 and CRYβB2P1 promote tumor growth, but their mechanisms for tumor promotion are likely distinct.ConclusionsOur findings provide novel data emphasizing the need to distinguish and study the biological effects of both CRYβB2 and CRYβB2P1 as both genes independently promote tumor progression. Our data demonstrate novel molecular mechanisms of two understudied, disparity-linked molecules.