Characterization of viral and human RNAs smaller than canonical MicroRNAs

Abstract

Recently identified small (20 to 40 bases) RNAs, such as microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) participate in important cellular pathways. In this report, we systematically characterized several novel features of human and viral RNA products smaller than miRNAs. We found that Kaposi sarcoma-associated herpesvirus K12-1 miRNA (23 bases) associates with a distinct, unusually small (17-base) RNA (usRNA) that can effectively downregulate a K12-1 miRNA target, human RAD21, suggesting that stable degradation-like products may also contribute to gene regulation. High-throughput sequencing reveals a diverse set of human miRNA-derived usRNAs and other non-miRNA-derived usRNAs. Human miRNA-derived usRNAs preferentially match to 5' ends of miRNAs and are also more likely to associate with the siRNA effector protein Ago2 than with Ago1. Many non-miRNA-derived usRNAs associate with Ago proteins and also frequently contain C-rich 3'-specific motifs that are overrepresented in comparison to Piwi-interacting RNAs and transcription start site-associated RNAs. We postulate that approximately 30% of usRNAs could have evolved to participate in biological processes, including gene silencing.

FIG. 1.

Detection of us-k12-1 and other putative small RNAs. (A) Northern blotting detected K12-1 (∼22 nt) and us-K12-1 (∼16 nt) in KSHV-positive cells (BCBL-1) but not in KSHV-negative cells (BJAB). Ethidium bromide staining (bottom) confirmed equal loading. (B) The number of sequence reads for various sizes of RNAs. Numerous usRNAs were also found in fraction B, likely due to RNA cross-hybridization and nonuniform RNA migration.

FIG. 2.

Characteristics of Ago-associated miRNA-derived usRNAs. (A) Ago-associated miRNA-derived usRNAs primarily match to 5′ positions 1 to 17 of miRNAs and rarely map to 3′ ends of miRNAs; percentage of miRNAs that match to usRNAs (17 to 18 nt) is plotted at each miRNA position. (B) Number of sequence reads associated with Ago1 and Ago2 and their ratios (Ago2 to Ago1) indicate the preferential association of Ago2 with 17- to 18-base-long miRNA-derived usRNAs. (C) Northern blot of Ago-associated K12-1 (top) and us-K12-1 (bottom) based on immunoprecipitation of Ago 1 to 4. Control, irrelevant antibody. The estimated Ago relative affinities with respect to K12-1 in binding us-K12-1 are indicated (see Materials and Methods). Relative affinities are normalized to that of Ago1.

FIG. 3.

Both miR-K12-1 and us-K12-1 can downregulate RAD21. (A) The 3′ UTR of the RAD21 mRNA contains two putative binding sites for K12-1 and us-K12-1 (underlined) at positions 3099 (site 1) and 3433 (site 2). (B) Immunoblots of endogenous RAD21 indicate that both miR-K12-1 and us-K12-1 downregulate the protein level of RAD21, in comparison to control RNA (Control). Tubulin levels confirm equal loading. For clarity, RAD21 levels are also normalized (Norm.) based on Tubulin signals (bottom). (C) Real-time RT-PCR of RAD21 transcripts demonstrate that miR-K12-1 and us-K12-1 also downregulate RAD21 mRNA levels. (D) Normalized firefly luciferase activity for WT or mutated (M1, M2, and M1 + 2) UTRs after transfection of K12-1 miRNA or us-K12-1. Note that although mutations in either binding site affect miR-K12-1 activity, us-K12-1 activity is largely unaffected by mutations in the canonical miRNA binding site (site 2). (E) Normalized firefly luciferase activity for WT UTR after transfection of either K12-1 miRNA or us-K12-1 at various concentrations (0.1 to 10 nM). All error bars represent standard error of the mean (n ≥ 3).

FIG. 4.

Ago-associated non-miRNA-derived sequences containing the 5′ and 3′ C-rich motifs. The sequences (17 to 18 nt) are highly conserved at the 5′ and 3′ end. The WebLogo representation (bottom) of the motif reveals several similarities with that of the CACCA-containing RNAs, particularly with respect to the strong preferences for C's at the 3′ end, C's at 5′ positions 4 and 5, and a subdued yet relevant preference for A in CACC at the 3′ end (see also Fig. 5).

FIG. 5.

The motifs CGCCA, CACCA, GCCGC, and GGGGG are preferentially located at specific 3′ positions of usRNAs and do not manifest such positional preferences in RNAs longer than 18 bases generally. The total number of RNAs (e.g., 23,438 piRNAs) used in the analysis and the total number of motif-containing RNAs (e.g., 313 CGCCA-containing RNAs) are indicated in parentheses.