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DNA replication occurs on mammalian chromosomes in a cell-type distinctive temporal order known as the replication timing program. We previously found that disruption of the noncanonical lncRNA genes ASAR6 and ASAR15 results in delayed replication timing and delayed mitotic chromosome condensation of human chromosome 6 and 15, respectively. ASAR6 and ASAR15 display random monoallelic expression, and display asynchronous replication between alleles that is coordinated with other random monoallelic genes on their respective chromosomes. Disruption of the expressed allele, but not the silent allele, of ASAR6 leads to delayed replication, activation of the previously silent alleles of linked monoallelic genes, and structural instability of human chromosome 6. In this report, we describe a second lncRNA gene (ASAR6-141) on human chromosome 6 that when disrupted results in delayed replication timing in cis. ASAR6-141 is subject to random monoallelic expression and asynchronous replication, and is expressed from the opposite chromosome 6 homolog as ASAR6. ASAR6-141 RNA, like ASAR6 and ASAR15 RNAs, contains a high L1 content and remains associated with the chromosome territory where it is transcribed. Three classes of cis-acting elements control proper chromosome function in mammals: origins of replication, centromeres; and telomeres, which are responsible for replication, segregation and stability of all chromosomes. Our work supports a fourth type of essential chromosomal element, “Inactivation/Stability Centers”, which express ASAR lncRNAs responsible for proper replication timing, monoallelic expression, and structural stability of each chromosome.Author summaryMammalian cells replicate their chromosomes during a highly ordered and cell type-specific program. Genetic studies have identified two long non-coding RNA genes, ASAR6 and ASAR15, as critical regulators of the replication timing program of human chromosomes 6 and 15, respectively. There are several unusual characteristics of the ASAR6 and ASAR15 RNAs that distinguish them from other long non-coding RNAs, including: being very long (>200 kb), lacking splicing of the transcripts, lacking polyadenylation, and being retained in the nucleus on the chromosomes where they are made. ASAR6 and ASAR15 also have the unusual property of being expressed from only one copy of the two genes located on homologous chromosome pairs. Using these unusual characteristics shared between ASAR6 and ASAR15, we have identified a second ASAR lncRNA gene located on human chromosome 6, which we have named ASAR6-141. ASAR6-141 is expressed from the opposite chromosome 6 homolog as ASAR6, and disruption of the expressed allele results in delayed replication of chromosome 6. ASAR6-141 RNA had previously been annotated as vlinc273. The very long intergenic non-coding (vlinc)RNAs represent a recently annotated class of RNAs that are long (>50 kb), non-spliced, and non-polyadenlyated nuclear RNAs. There are currently >2,700 vlincRNAs expressed from every chromosome, are encoded by >15% of the human genome, and with a few exceptions have no known function. Our results suggest the intriguing possibility that the vlinc class of RNAs may be functioning to control the replication timing program of all human chromosomes.

The human DNA mismatch repair gene homologue hMSH2, on chromosome 2p is involved in hereditary non-polyposis colon cancer (HNPCC). On the basis of linkage data, a second HNPCC locus was assigned to chromosome 3p21-23 (ref. 3). Here we report that a human gene encoding a protein, hMLH1 (human MutL homologue), homologous to the bacterial DNA mismatch repair protein MutL, is located on human chromosome 3p21.3-23. We propose that hMLH1 is the HNPCC gene located on 3p because of the similarity of the hMLH1 gene product to the yeast DNA mismatch repair protein, MLH1, the coincident location of the hMLH1 gene and the HNPCC locus on chromosome 3, and hMLH1 missense mutations in affected individuals from a chromosome 3-linked HNPCC family.