SNOntology: Myriads of novel snornas or just a mirage?

<p>Abstract</p> <p>Background</p> <p>Small nucleolar RNAs (snoRNAs) are a large group of non-coding RNAs (ncRNAs) that mainly guide 2'-O-methylation (C/D RNAs) and pseudouridylation (H/ACA RNAs) of ribosomal RNAs. The pattern of rRNA modifications and the set of snoRNAs that guide these modifications are conserved in vertebrates. Nearly all snoRNA genes in vertebrates are localized in introns of other genes and are processed from pre-mRNAs. Thus, the same promoter is used for the transcription of snoRNAs and host genes.</p> <p>Results</p> <p>The series of studies by Dahai Zhu and coworkers on snoRNAs and their genes were critically considered. We present evidence that dozens of species-specific snoRNAs that they described in vertebrates are experimental artifacts resulting from the improper use of Northern hybridization. The snoRNA genes with putative intrinsic promoters that were supposed to be transcribed independently proved to contain numerous substitutions and are, most likely, pseudogenes. In some cases, they are localized within introns of overlooked host genes. Finally, an increased number of snoRNA genes in mammalian genomes described by Zhu and coworkers is also an artifact resulting from two mistakes. First, numerous mammalian snoRNA pseudogenes were considered as genes, whereas most of them are localized outside of host genes and contain substitutions that question their functionality. Second, Zhu and coworkers failed to identify many snoRNA genes in non-mammalian species. As an illustration, we present 1352 C/D snoRNA genes that we have identified and annotated in vertebrates.</p> <p>Conclusions</p> <p>Our results demonstrate that conclusions based only on databases with automatically annotated ncRNAs can be erroneous. Special investigations aimed to distinguish true RNA genes from their pseudogenes should be done. Zhu and coworkers, as well as most other groups studying vertebrate snoRNAs, give new names to newly described homologs of human snoRNAs, which significantly complicates comparison between different species. It seems necessary to develop a uniform nomenclature for homologs of human snoRNAs in other vertebrates, e.g., human gene names prefixed with several-letter code denoting the vertebrate species.</p

Author&apos;s personal copy 5′-flanking sequences can dramatically influence 4.5SH RNA gene transcription by RNA-polymerase III

Received by S.M. Mirkin Keywords: Small RNA 4.5S RNA H 4.5S RNA Internal promoter Mus musculus 4.5SH RNA is a 94 nt small nuclear RNA with an unknown function. Hundreds of its genes are present in the genomes of rodents of six families including Muridae. 4.5SH RNA genes contain an internal RNA-polymerase III promoter consisting of A and B boxes. Here we studied the influence of 5′-flanking sequences on the transcription of a mouse 4.5SH RNA gene. We found that replacement of the upstream sequence can dramatically change the 4.5SH RNA gene transcription efficiency. Various DNA fragments inserted immediately upstream from 4.5SH RNA gene completely inhibited its in vitro transcription, whereas others promoted it. The shortening of the native mouse 5′-flanking sequence of 4.5SH RNA gene to 42 bp resulted in the activation of an additional illegal transcription start site in upstream region. Transcription of the 4.5SH RNA gene with various upstream sequences in transfected HeLa cells revealed the differences between the tests performed in vivo and in vitro: in whole cells, only the construct with 5′-flanking native sequence could be transcribed. Apparently, at least some regions of the native 5′-flanking sequence of 4.5SH RNA genes have been selected during evolution for high transcription activity

Wide distribution of short interspersed elements among eukaryotic genomes

AbstractMost short interspersed elements (SINEs) in eukaryotic genomes originate from tRNA and have internal promoters for RNA polymerase III. The promoter contains two boxes (A and B) spaced by ∼33 bp. We used oligonucleotide primers specific to these boxes to detect SINEs in the genomic DNA by polymerase chain reaction (PCR). Appropriate DNA fragments were revealed by PCR in 30 out of 35 eukaryotic species suggesting the wide distribution of SINEs. The PCR products were used for hybridization screening of genomic libraries which resulted in identification of four novel SINE families. The application of this approach is illustrated by discovery of a SINE family in the genome of the bat Myotis daubentoni. Members of this SINE family termed VES have an additional B-like box, a putative polyadenylation signal and RNA polymerase III terminator

Transcripts synthesized by RNA polymerase III can be polyadenylated in an AAUAAA-dependent manner

It is well known that nearly all eukaryotic mRNAs contain a 3′ poly(A) tail. A polyadenylation signal (AAUAAA) nearby the 3′ end of pre-mRNA is required for poly(A) synthesis. The protein complex involved in the pre-mRNA polyadenylation is coupled with RNA polymerase II during the transcription of a gene. According to the commonly accepted view, only RNAs synthesized by RNA polymerase II can be polyadenylated in an AAUAAA-dependent manner. Here we report the polyadenylation of short interspersed elements (SINEs) B2 and VES transcripts generated by RNA polymerase III. HeLa cells were transfected with SINE constructs with or without polyadenylation signals. The analyses of the SINE transcripts showed that only the RNAs with the AAUAAA-signal contained poly(A) tails. Polyadenylated B2 RNA was found to be much more stable in cells than B2 RNA without a poly(A) tail

Complementarity of end regions increases the lifetime of small RNAs in mammalian cells.

Two RNAs (4.5SH and 4.5SI) with unknown functions share a number of features: short length (about 100 nt), transcription by RNA polymerase III, predominately nuclear localization, the presence in various tissues, and relatively narrow taxonomic distribution (4 and 3 rodent families, respectively). It was reported that 4.5SH RNA turns over rapidly, whereas 4.5SI RNA is stable in the cell, but their lifetimes remained unknown. We showed that 4.5SH is indeed short-lived (t(1/2)~18 min) and 4.5SI is long-lived (t(1/2)~22 h) in Krebs ascites carcinoma cells. The RNA structures specifying rapid or slow decay of different small cellular RNAs remain unstudied. We searched for RNA structural features that determine the short lifetime of 4.5SH in comparison with the long lifetime of 4.5SI RNA. The sequences of genes of 4.5SH and 4.5SI RNAs were altered and human cells (HeLa) were transfected with these genes. The decay rate of the original and altered RNAs was measured. The complementarity of 16-nt end regions of 4.5SI RNA proved to contribute to its stability in cells, whereas the lack of such complementarity in 4.5SH RNA caused its rapid decay. Possible mechanisms of the phenomenon are discussed

Ere, a Family of Short Interspersed Elements in the Genomes of Odd-Toed Ungulates (Perissodactyla)

Short Interspersed Elements (SINEs) are eukaryotic retrotransposons transcribed by RNA polymerase III (pol III). Many mammalian SINEs (T+ SINEs) contain a polyadenylation signal (AATAAA), a pol III transcription terminator, and an A-rich tail in their 3′-end. The RNAs of such SINEs have the capacity for AAUAAA-dependent polyadenylation, which is unique to pol III-generated transcripts. The structure, evolution, and polyadenylation of the Ere SINE of ungulates (horses, rhinos, and tapirs) were investigated in this study. A bioinformatics analysis revealed the presence of up to ~4 × 105 Ere copies in representatives of all three families. These copies can be classified into two large subfamilies, EreA and EreB, the former distinguished by an additional 60 bp sequence. The 3′-end of numerous EreA and all EreB copies exhibit a 50 bp sequence designated as a terminal domain (TD). The Ere family can be further subdivided into subfamilies EreA_0TD, EreA_1TD, EreB_1TD, and EreB_2TD, depending on the presence and number of terminal domains (TDs). Only EreA_0TD copies can be assigned to T+ SINEs as they contain the AATAAA signal and the TCTTT transcription terminator. The analysis of young Ere copies identified by comparison with related perissodactyl genomes revealed that EreA_0TD and, to a much lesser extent, EreB_2TD have retained retrotranspositional activity in the recent evolution of equids and rhinoceroses. The targeted mutagenesis and transfection of HeLa cells were used to identify sequences in equine EreA_0TD that are critical for the polyadenylation of its pol III transcripts. In addition to AATAAA and the transcription terminator, two sites in the 3′ half of EreA, termed the β and τ signals, were found to be essential for this process. The evolution of Ere, with a particular focus on the emergence of T+ SINEs, as well as the polyadenylation signals are discussed in comparison with other T+ SINEs

Tail Wags Dog&rsquo;s SINE: Retropositional Mechanisms of Can SINE Depend on Its A-Tail Structure

SINEs, non-autonomous short retrotransposons, are widespread in mammalian genomes. Their transcripts are generated by RNA polymerase III (pol III). Transcripts of certain SINEs can be polyadenylated, which requires polyadenylation and pol III termination signals in their sequences. Our sequence analysis divided Can SINEs in canids into four subfamilies, older a1 and a2 and younger b1 and b2. Can_b2 and to a lesser extent Can_b1 remained retrotranspositionally active, while the amplification of Can_a1 and Can_a2 ceased long ago. An extraordinarily high Can amplification was revealed in different dog breeds. Functional polyadenylation signals were analyzed in Can subfamilies, particularly in fractions of recently amplified, i.e., active copies. The transcription of various Can constructs transfected into HeLa cells proposed AATAAA and (TC)n as functional polyadenylation signals. Our analysis indicates that older Can subfamilies (a1, a2, and b1) with an active transcription terminator were amplified by the T+ mechanism (with polyadenylation of pol III transcripts). In the currently active Can_b2 subfamily, the amplification mechanisms with (T+) and without the polyadenylation of pol III transcripts (T&minus;) irregularly alternate. The active transcription terminator tends to shorten, which renders it nonfunctional and favors a switch to the T&minus; retrotransposition. The activity of a truncated terminator is occasionally restored by its elongation, which rehabilitates the T+ retrotransposition for a particular SINE copy

Analysis of SINE Families B2, Dip, and Ves with Special Reference to Polyadenylation Signals and Transcription Terminators

Short Interspersed Elements (SINEs) are eukaryotic non-autonomous retrotransposons transcribed by RNA polymerase III (pol III). The 3′-terminus of many mammalian SINEs has a polyadenylation signal (AATAAA), pol III transcription terminator, and A-rich tail. The RNAs of such SINEs can be polyadenylated, which is unique for pol III transcripts. Here, B2 (mice and related rodents), Dip (jerboas), and Ves (vespertilionid bats) SINE families were thoroughly studied. They were divided into subfamilies reliably distinguished by relatively long indels. The age of SINE subfamilies can be estimated, which allows us to reconstruct their evolution. The youngest and most active variants of SINE subfamilies were given special attention. The shortest pol III transcription terminators are TCTTT (B2), TATTT (Ves and Dip), and the rarer TTTT. The last nucleotide of the terminator is often not transcribed; accordingly, the truncated terminator of its descendant becomes nonfunctional. The incidence of complete transcription of the TCTTT terminator is twice higher compared to TTTT and thus functional terminators are more likely preserved in daughter SINE copies. Young copies have long poly(A) tails; however, they gradually shorten in host generations. Unexpectedly, the tail shortening below A10 increases the incidence of terminator elongation by Ts thus restoring its efficiency. This process can be critical for the maintenance of SINE activity in the genome