Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Sep 21:14:23.
doi: 10.1186/1471-2199-14-23.

3' terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing

Katherine C Goldfarb  1 , Thomas R Cech
Affiliations

3' terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing

Katherine C Goldfarb et al. BMC Mol Biol. .

Abstract

Background: Post-transcriptional 3' end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3' RACE coupled with high-throughput sequencing to characterize the 3' terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes.

Results: The 3' terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3' terminus of an in vitro transcribed MRP RNA control and the differing 3' terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR).

Conclusions: 3' RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3' terminal sequences of noncoding RNAs.

PubMed Disclaimer

Figures

Figure 1
Figure 1
3′ RACE with deep sequencing, schematic of method used. a Ligation of whole cell RNA with 5′ degenerate indexed appendices (orange), b Reverse transcription with appendix-specific primer, c 3′ RACE-PCR selection with indexed (black and white barcode) gene-specific F primer and universal R primer containing Illumina adapter sequences (blue), d Library amplification with general primers to the adapter sequences, e High-throughput insert and RACE index sequencing on Illumina MiSeq (sequencing primers in purple), f Bioinformatic trimming of reads to analyze 3′ terminal sequences (yellow). See Methods for detailed protocol.
Figure 2
Figure 2
RACE sequencing reveals a distribution of 3′ termini for MRP RNA. a The majority of HEK293T cellular MRP RNA 3′ ends are genomically encoded (black pie slice), with a modest portion of these containing oligo(A)n additions to these genomically encoded ends (range of n=1-10, light blue slice). In contrast, while nearly three quarters of the reads from in vitro transcribed MRP (ivt-MRP) are the exact target sequence (grey slice), the remaining reads consist of single nucleotide additions to this designed end (+A in blue; +C in green; +G in yellow; +U in red). Both cellular and ivt-MRP displayed a small portion of other simple sequences shown as white slices (i.e. …CUCC, …CUGC, and see Figure 3). Total trimmed reads = 1,497,440 (cellular) and 906,145 (ivt). b Reads from endogenous MRP terminate at specific nucleotides in the flanking RMRP gene with non-random abundances (black bars) and distinct probabilities of oligo(A) addition (light blue caps). As an example, the relative frequency of different numbers (n) of A’s found on one of the genomically encoded termini is illustrated in the inset. The major genomically encoded end is not observed on ivt-MRP (lower panel), nor is there appreciable propensity for oligo(A) addition. Although a single A is sometimes added to the designed terminus, C and G are added with similar frequency.
Figure 3
Figure 3
Loopback extensions on the ivt-MRP 3′ end are observed at low abundance by deep sequencing. a Approximately one per cent of observed 3′ ends contain complex sequences that are not explained by the in vitro template (violet slice). The majority of the reads from in vitro transcribed MRP (ivt-MRP) are the exact target sequence (grey slice), while the remainder can be categorized as single nucleotide additions to this designed end (+A in blue; +C in green; +G in yellow; +U in red), or other simple sequences (i.e. …CUCC, …CUGC, white slice). b Examples of possible duplex registers to template the observed sequences of loopback extensions.
Figure 4
Figure 4
Other noncoding RNAs display 3′ end profiles somewhat different from those of endogenous MRP RNA. a Categorized reads and b genomically encoded/oligo(A) addition profiles are shown for RNase P RNA, U3 snoRNA and the RNA component of human telomerase, hTR. Genomically encoded termini are in black, oligo(A)n additions to genomically encoded ends are in blue, other sequences in white. The top sequence listed for each RNA is the annotated 3′ terminus. A representative probability distribution for the number (n) of A’s is included for hTR in inset. Total trimmed reads = 353,190 (RNase P); 246,485 (U3 snoRNA); and 1,013 (hTR).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jones MR, Quinton LJ, Blahna MT, Neilson JR, Fu S, Ivanov AR, Wolf DA, Mizgerd JP. Zcchc11-dependent uridylation of microRNA directs cytokine expression. Nat Cell Biol. 2009;11:1157–1163. - PMC - PubMed
    1. Chiang HR, Schoenfeld LW, Ruby JG, Auyeung VC, Spies N, Baek D, Johnston WK, Russ C, Luo S, Babiarz JE. et al.Mammalian microRNAs: experimental evaluation of novel and previously annotated genes. Genes Dev. 2010;24:992–1009. - PMC - PubMed
    1. Heo I, Ha M, Lim J, Yoon MJ, Park JE, Kwon SC, Chang H, Kim VN. Mono-uridylation of pre-microRNA as a key step in the biogenesis of group II let-7 microRNAs. Cell. 2012;151:521–532. - PubMed
    1. Choi YS, Patena W, Leavitt AD, McManus MT. Widespread RNA 3′-end oligouridylation in mammals. RNA. 2012;18:394–401. - PMC - PubMed
    1. Chen Y, Sinha K, Perumal K, Reddy R. Effect of 3′ terminal adenylic acid residue on the uridylation of human small RNAs in vitro and in frog oocytes. RNA. 2000;6:1277–1288. - PMC - PubMed

Publication types