Most of the eukaryotic genome is transcribed, yielding a complex repertoire of RNAs that includes tens of thousands of noncoding RNAs with little or no predicted protein-coding capacity. Among these are well-studied small RNAs, such as microRNAs, as well as many other classes of small and long transcripts whose functions and mechanisms of biogenesis are less clear – but likely no less important, especially since some are associated with diseases such as cancer and developmental disorders.

Our goal is to characterize the mechanisms by which these poorly characterized noncoding RNAs are generated, regulated, and function, thereby revealing novel insights into RNA biology and developing new methods to treat diseases.

RESEARCH TOPICS:

Regulation of MALAT1, MEN β, and other non-polyadenylated RNAs

Circular RNAs: Unexpected outputs of protein-coding genes

(1) Regulation of MALAT1, MEN β, and other non-polyadenylated RNAs

Much of our work has focused on the MALAT1 locus, which is over-expressed in many human cancers and produces a long nuclear-retained noncoding RNA as well as a tRNA-like small RNA known as mascRNA, MALAT1-associated small cytoplasmic RNA. Despite being an RNA polymerase II transcript, the 3’ end of MALAT1 is produced not by canonical cleavage/polyadenylation but instead by recognition and cleavage of the tRNA-like structure by RNase P (Figure 1).

MALAT1

Figure 1. Non-canonical 3’ end processing of MALAT1

The MEN β long nuclear-retained noncoding RNA, also known as NEAT1_2, is similarly processed at its 3’ end by RNase P. Surprisingly, although processing of MEN β produces a tRNA-like small RNA, it is generally rapidly degraded in vivo. Pursuing this observation, we revealed a universally conserved quality control mechanism mediated by the CCA-adding enzyme (Figure 2). Normally, the CCA-adding enzyme adds CCA to the 3’ ends of tRNAs, a critical step in tRNA biogenesis that generates the amino acid attachment site. However, the enzyme adds CCACCA to structurally unstable tRNAs and tRNA-like small RNAs (including the MEN β tRNA-like transcript and certain hypomodified tRNAs) marking them for degradation. We conjecture that CCACCA addition prevents errors in translation and controls tRNA levels, an especially critical function considering that even slight changes in tRNA levels can drive cell proliferation and oncogenic transformation.

mismatch in acceptor system

Figure 2. A mismatch in the acceptor stem of the MEN β tRNA-like small RNA triggers CCACCA addition
From Wilusz et al. (2011) Science 334: 817-821.

As the mature 3’ ends of MALAT1 and MEN β are generated by RNase P and not by the cleavage and polyadenylation machinery, these long noncoding RNAs lack poly(A) tails. Nevertheless, these RNAs are expressed at levels higher than many protein-coding genes. We showed that the 3’ ends of these noncoding RNAs are protected from 3’-5’ exonucleases by highly conserved triple helical structures (Figure 3).

MALAT1 and MEN beta ends

Figure 3. A triple helix stabilizes the 3’ ends of MALAT1 and MEN β
From Wilusz et al. (2012) Genes Dev 26: 2392-2407.

Surprisingly, when these triple helical structures are placed downstream from an open reading frame, the transcript is efficiently translated in vivo despite the lack of a poly(A) tail. This result challenges the common paradigm that long poly(A) tails are required for efficient protein synthesis and suggests that non-polyadenylated RNAs may produce functional peptides in vivo via mechanisms that are likely independent of poly(A) binding protein. To address these issues, we are currently elucidating the molecular mechanism by which a triple helix functions as a translational enhancer. In addition, we are developing approaches to identify additional triple helices that form across the transcriptome, thereby revealing new paradigms for how RNA structures regulate gene expression.

KEY PUBLICATIONS
  • Doucet, A.J., Wilusz, J.E., Miyoshi, T., Liu, Y., and Moran, J.V. (2015) A 3’ poly(A) tract is required for LINE-1 retrotransposition. Mol Cell 60:728-741.
  • Kuhn, C.-D., Wilusz, J.E., Zheng, Y., Beal, P.A., and Joshua-Tor, L. (2015) On-enzyme refolding permits small RNA and tRNA surveillance by the CCA-adding enzyme. Cell 160:644-658.
  • Wilusz, J.E., JnBaptiste, C.K., Lu, L.Y., Kuhn, C.-D., Joshua-Tor, L., and Sharp, P.A. (2012) A triple helix stabilizes the 3’ ends of long noncoding RNAs that lack poly(A) tails. Genes Dev. 26:2392-2407.
  • Wilusz, J.E., Whipple, J.M., Phizicky, E.M., and Sharp, P.A. (2011) tRNAs marked with CCACCA are targeted for degradation. Science 334:817-821.
  • Wilusz, J.E. and Spector, D.L. (2010) An unexpected ending: non-canonical 3’ end processing mechanisms. RNA 16:259-266.
  • Sunwoo, H., Dinger, M.E., Wilusz, J.E., Amaral, P.P., Mattick, J.S., and Spector, D.L. (2009) MEN ε/β nuclear-retained non-coding RNAs are up-regulated upon muscle differentiation and are essential components of paraspeckles. Genome Res. 19:347-359.
  • Wilusz, J.E., Freier, S.M., and Spector, D.L. (2008) 3’ end processing of a long nuclear-retained noncoding RNA yields a tRNA-like cytoplasmic RNA. Cell 135:919-932.

(2) Circular RNAs: Unexpected outputs of protein-coding genes

Deep sequencing has revealed thousands of eukaryotic protein-coding genes that defy the central dogma, producing circular noncoding RNAs rather than linear messenger RNAs. For some genes, the abundance of the circular RNA exceeds that of the associated linear mRNA by a factor of 10, raising the interesting possibility that the function of some protein-coding genes may actually be to produce circular noncoding RNAs, not proteins. These circular RNAs are generated when the pre-mRNA splicing machinery “backsplices” and joins a splice donor to an upstream splice acceptor (Figure 4). Once produced, it is largely unclear what circular RNAs do, although two are known to efficiently modulate the activity of microRNAs.

circular RNAs

Figure 4. Backsplicing produces circular RNAs, some of which function as microRNA sponges
            From Wilusz and Sharp (2013) Science 340: 440-441.

With the exception of the first and last exons of genes, every other exon in the genome has splicing signals at its 5’ and 3’ ends and theoretically can circularize. However, every exon does not circularize, and, in some cases, multiple exons are present in a circular RNA. We recently showed that repetitive elements, e.g. SINE elements, in the flanking introns are critical determinants of whether the intervening exon(s) circularize. When repeat sequences from the flanking introns base pair to one another, the splice sites are brought into close proximity and backsplicing occurs (Figure 5). Although mechanistically simple, this step occurs in a highly selective manner, as the sequence of the repeats can drastically alter the efficiency of circular RNA production. We additionally showed that circular RNA levels can be controlled by multiple hnRNP and SR proteins acting in a combinatorial manner.

circular RNA production

Figure 5. Short intronic repeats facilitate circular RNA production
            From Liang and Wilusz (2014) Genes Dev 28: 2233-2247.

We are continuing to elucidate the mechanism by which circular RNAs are produced. In particular, we are interested in determining how cellular cues can alter the ratio of linear mRNA to circular RNA for a given gene. We are also focused on identifying biological functions for circular RNAs, thereby revealing novel insights into how circular RNAs fit into the regulatory landscape of the cell.

KEY PUBLICATIONS

Kramer, M.C., Liang, D., Tatomer, D.C., Gold, B., Cherry, S., and Wilusz, J.E. (2015) Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins. Genes Dev 29:2168-2182.

  • Liang, D. and Wilusz, J.E. (2014) Short intronic repeat sequences facilitate circular RNA production. Genes Dev. 28:2233-2247.
  • Wilusz, J.E. and Sharp, P.A. (2013) A circuitous route to noncoding RNA. Science 340:440-441.