No Proteins, No Problem: Viroids Cause Disease By Silencing RNA

(Posted on Wednesday, March 15, 2023)

This article is part of a series on viroids. The first, which can be read here, gave an overview of what viroids are and how they replicate. The second article described the plant host range of viroids and their economic impact.

Viroids are small, circular slivers of ribonucleic acid (RNA) that do not encode any proteins. Despite this, they can self-replicate and damage hosts. Think of proteins as tools: they have specific functions that help achieve some end goal. Most viruses, for example, make use of proteins not only to enter host cells, but also to replicate once inside. Some of the nastier viruses also use proteins to evade or even suppress host immune responses. How do viroids cause disease without any help from proteins? Although the exact mechanisms of damage remain elusive, a process known as RNA interference (RNAi) is thought to play a central role. 

What is RNA Interference?

Many plants and animals use RNA interference (RNAi) as a means of regulating gene expression — it helps them switch certain genes, and by extension proteins, “off.” Genes encode information, and when genes are expressed, this information is turned into a functional product with a specific purpose. Modulating which genes are expressed, and when they are expressed, helps organisms keep their vital functions in balance. For example, gene expression of insulin increases following a meal in response to a spike in blood sugar; insulin acts as a key that allows blood sugar to enter our cells, where it can be used as energy. Depending on the meal, more or less insulin may be produced. 

Gene expression is also vital to successful immune responses. During infection, the host immune system is constantly upregulating —increasing the expression of— and downregulating —decreasing the expression of— a variety of different proteins. These proteins contribute to inflammation, help direct immune cells to where they need to be, and so on. But this is a very delicate dance; too much inflammation can be just as damaging as too little inflammation, for instance. So organisms are constantly modulating gene expression in order to keep things “just right”. 

RNA interference, as mentioned, is one mechanism of gene expression. How does it work? Broadly speaking, small snippets of RNA shut down protein synthesis by binding to, and interfering with, the messenger RNAs that code for those proteins. The slivers of RNA that enable the gene silencing can be broken down into two classes: microRNA (miRNA) and small interfering RNA (siRNA). Both of these small RNA types are usually between 20 and 24 nucleotides long — tiny. For comparison, SARS-CoV-2 is made up of roughly 30,000 nucleotides. MicroRNAs are derived from a double-stranded region of a hairpin-shaped precursor (Figure 1), whereas small interfering RNAs are generated from longer regions of double-stranded RNA (dsRNA). 

FIGURE 1. Examples of hairpin-shaped miRNA precursors, with the mature miRNAs shown in red. SOURCE: Wikipedia

Despite the subtle differences between the two types, the general pathway through which microRNAs and small interfering RNAs induce gene silencing remains the same. First, their precursors —pre-microRNA and double-stranded RNA, respectively — are ferried from the nucleus into the cytoplasm of the cell. Once there, the precursors are cut into siRNAs or miRNAs by a protein called Dicer. Next, the small RNA pieces are incorporated into something called the RNA-induced silencing complex (RISC). RISC uses miRNA or siRNA as a template and begins to search for a complementary strand of messenger RNA. After recognizing and binding to the complementary messenger RNA, the complex can interfere with gene expression in two ways: it either uses a protein called Argonaute to “slice up” the mRNA, after which it gets degraded by the cell, or it simply inhibits mRNA from being translated by physically blocking the machinery used to do so (Figure 2).

FIGURE 2.  A schematic diagram of RNA interference, either through cleavage of messenger RNA (right) or through physical inhibition (left). SOURCE: National Library of Medicine, NIH

MicroRNAs generally do not perfectly match up to messenger RNA strands; because they are not specific to any one mRNA, they can inhibit many different, sequentially-similar messenger RNAs. In contrast, small interfering RNAs are highly specific, each binding to and silencing only one particular messenger RNA target. Regardless, both successfully silence gene expression.

Viroids: Gene Silencing Through Mimicry? 

Immune systems and pathogens are involved in a constant, ongoing chess match: immune systems are exposed to pathogens, develop mechanisms to block and destroy them, and the pathogens, in turn, “learn” to evade these mechanisms. Sometimes, pathogens manage to use those selfsame defense mechanisms to their advantage. Although RNA interference is generally used by plants to target and suppress foreign RNA, viroids may in fact be hijacking the process, using it to instead damage their hosts. 

All of this begins with a case of mistaken identity. Recall that RNA interference makes use of small interfering RNAs and microRNAs, both of which are derived from precursor double-stranded RNA. During replication, viroids depend on an intermediate step where their RNA briefly takes on a double-stranded structure. As such, the viroid RNA may be erroneously recognized by Dicer as a precursor and chopped up into small fragments, called viroid short RNAs (vsRNAs). Given their similarity to the small RNAs used for gene silencing, viroid short RNAs may get incorporated into RNA-induced silencing complex (RISC) and, from there, interfere with host gene expression (Figure 3). 

FIGURE 3. Cutting of precursor microRNA (pre-miRNA) by the nucleases Dicer or Drosha releases the small regulatory RNAs—miRNA. Cutting of viroid RNA by the same nucleases releases small RNA molecules—vsRNA. These mimic the action of miRNA. The RISC complex helps both vsRNA and miRNA to bind to complementary sequences on target messenger RNAs. This alters the expression of the mRNA. In the case of the viroid, expression is abnormal and disease results. FROM: “Viruses, Viroids, and Prions” CLARK ET AL. 2019

Evidence for viroid-induced RNA interference comes from a number of sources. For one, deep sequencing of viroid short RNAs from tomato plants infected with potato spindle tuber viroid (PSTVd) has shown that most viroid short RNAs are derived from a part of the viroid known to be related to disease symptoms, called the pathogenicity domain. Another study found that proteins associated with disease resistance in plants —serine/threonine kinases— were negatively affected by viroid infection. Importantly, exposing plants to just the viroid short RNAs, without the rest of the viroid, was enough to cause damage similar to that seen during infection. Similarly, researchers have found that infection with the tomato planta macho viroid (TPMVd) leads to the downregulation of gene networks associated with plant development and growth.

Although additional mechanisms of viroid-induced disease may still surface, current evidence suggests RNA interference, and the subsequent downregulation of host genes, plays a central role.

The next article in this series will take a closer look at ribozymes, which are slivers of RNA that, despite not encoding a protein, can act as enzymes. This is the closest thing viroids have to proteins.