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Automated genome annotation is essential for extracting biological information from sequence data. The identification and annotation of tRNA genes is frequently performed by the software package tRNAscan-SE, the output of which is listed for selected genomes in the Genomic tRNA database (GtRNAdb). Here, we highlight a pervasive error in prokaryotic tRNA gene sets on GtRNAdb: the mis-categorization of partial, non-canonical tRNA genes as standard, canonical tRNA genes. Firstly, we demonstrate the issue using the tRNA gene sets of 20 organisms from the archaeal taxon Thermococcaceae. According to GtRNAdb, these organisms collectively deviate from the expected set of tRNA genes in 15 instances, including the listing of eleven putative canonical tRNA genes. However, after detailed manual annotation, only one of these eleven remains; the others are either partial, non-canonical tRNA genes resulting from the integration of genetic elements or CRISPR-Cas activity (seven instances), or attributable to ambiguities in input sequences (three instances). Secondly, we show that similar examples of the mis-categorization of predicted tRNA sequences occur throughout the prokaryotic sections of GtRNAdb. While both canonical and non-canonical prokaryotic tRNA gene sequences identified by tRNAscan-SE are biologically interesting, the challenge of reliably distinguishing between them remains. We recommend employing a combination of (i) screening input sequences for the genetic elements typically associated with non-canonical tRNA genes, and ambiguities, (ii) activating the tRNAscan-SE automated pseudogene detection function, and (iii) scrutinizing predicted tRNA genes with low isotype scores. These measures greatly reduce manual annotation efforts, and lead to improved prokaryotic tRNA gene set predictions.

The RB1 gene is the first described human tumor suppressor gene and plays an integral role in the development of retinoblastoma, a pediatric malignancy of the eye. Since its discovery, the stepwise characterization and cloning of RB1 have laid the foundation for numerous advances in the understanding of tumor suppressor genes, retinoblastoma tumorigenesis, and inheritance. Knowledge of RB1 led to a paradigm shift in the field of cancer genetics, including widespread acceptance of the concept of tumor suppressor genes, and has provided crucial diagnostic and prognostic information through genetic testing for patients affected by retinoblastoma. This article reviews the long history of RB1 gene research, characterization, and cloning, and also discusses recent advances in retinoblastoma genetics that have grown out of this foundational work.

DNA hypermethylation events occur frequently in human cancers, but less is known of the mechanisms leading to their initiation. Retinoblastoma, an intraocular cancer affecting young children, involves bi-allelic inactivation of the RB1 gene (RB−/-). RB1 encodes a tumour suppressing, cell cycle regulating transcription factor (pRB) that binds and regulates the RB1 core and other E2F responsive promoters with epigenetic functions that include recruitment of histone deacetylases (HDACs). Evidence suggests that bi-allelic epigenetic inactivation/hypermethylation of the RB1 core promoter (PrE-/E-), is specific to sporadic retinoblastomas (frequency~10%), whereas heritable RB1 promoter variants (Pr−/+, frequency~1-2%) are not associated with known epigenetic phenomena. We report heritable Pr−/- retinoblastomas with the expected loss of pRB expression, in which hypermethylation consistent with distal boundary displacement (BD) relative to normal peripheral blood DNAs was detected in 4/4 cases. In contrast, proximal BD was identified in 16/16 RB−/- retinoblastomas while multiple boundaries distal of the core promoter was further identified in PrE-/E-and PrE-/E+ retinoblastomas. However, weak or no DNA hypermethylation/BD in peripheral blood DNA was detected in 8/9 Pr−/+ patients, with the exception, a carrier of a microdeletion encompassing several RB1 promoter elements. These findings suggest that loss of boundary control may be a critical step leading to epigenetic inactivation of the RB1 gene and that novel DNA methylation boundaries/profiles identified in the RB1 promoter of Pr−/- retinoblastomas, may be the result of epigenetic phenomena associated with epimutation in conjunction with loss of pRB expression/binding and/or RB1 promoter interactions with boundary control elements.

CDH11 gene copy number and expression are frequently lost in human retinoblastomas and in retinoblastomas arising in TAg-RB mice. To determine the effect of Cdh11 loss in tumorigenesis, we crossed Cdh11 null mice with TAg-RB mice. Loss of Cdh11 had no gross morphological effect on the developing retina of Cdh11 knockout mice, but led to larger retinal volumes in mice crossed with TAg-RB mice (p = 0.01). Mice null for Cdh11 presented with fewer TAg-positive cells at postnatal day 8 (PND8) (p = 0.01) and had fewer multifocal tumors at PND28 (p = 0.016), compared to mice with normal Cdh11 alleles. However, tumor growth was faster in Cdh11-null mice between PND8 and PND84 (p = 0.003). In tumors of Cdh11-null mice, cell death was decreased 5- to 10-fold (p