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Chapter 2: RNPs, small RNAs, miRNAs

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Key points

RNA is processed into smaller RNA fragments that can have functions

Short RNAs appear to be crucial for gene regulation

RNAs are (almost?) always associated with proteins, forming
RNA:protein complexes

RNA functions range from catalytic activity to pure structural

scaffolding functions




The

Complete sequencing of the human genome revealed that only about 2% of the genetic
information is covered by protein coding genes
[1]
.
Interestingly, less complex eukaryotes
encode for a similar number of proteins. Therefore, it is very likely that non
-
protein coding
genomic regions contribute significantly to the complexity of higher eukaryotes. Gene
expression analyses revealed that active transcription is not restricted to protein
-
coding genes
but includes different classes of non
-
protein
-
coding RNAs. It is further believed that about
90% of the human genome is permanently transcribed giving rise to
a plethora of non
-
coding
transcripts
[2]
.
Such non
-
coding RNAs play impor
tant roles in many cellular processes
including transcription, pre
-
mRNA processing or translation. Several classes of non
-
coding
RNAs exist and some of them are well studied including small RNAs that are essential for
mRNA maturation processes or translati
on. A class of small non
-
coding RNAs, which has
been discovered more recently, is formed by so
-
called small regulatory RNAs. These small
RNAs include short interfering RNAs (siRNAs) or microRNAs are function as fundamental
regulators of gene expression.
Th
e regulatory functions of the small RNAs can be seen as an
echo of the previous RNA world that was discussed in chapter 1.

However, non
-
coding RNAs do not function on their own but are incorporated into large
protein complexes, generally referred to as rib
onucleoprotein particles or ribonucleoproteins
(RNPs). RNPs are typically composed of a proteinous component and a RNA component.
Their functionality ranges from true ribozymes with the RNA as the catalytic center to large
ribonucleoprotein complexes or pa
rticles where the RNA serves as scaffold for complex
formation and function. In both cases, the non
-
coding RNA adopts characteristic and highly
complex secondary structures. The composition of RNPs ranges from one RNA molecule
with one single protein to hu
ge macromolecular structures with several RNAs associated with
hundreds of protein components. The ribosome and the spliceosome are the most prominent
examples for such macromolecular complexes (
see chapter 3
). However, it is important to
emphasize that in

any RNP both RNA and proteins cooperate to fulfill their specific
biological functions.

In eukaryotic cells, non
-
coding RNA
-
containing RNPs are required for mRNA maturation as
well as transcription and translation. Small regulatory RNAs can regulate trans
cription in a
number of different organisms. U
-
rich small nuclear RNPs (U snRNPs) are the major
constituents of the spliceosome, which removes introns from pre
-
mRNAs during splicing.
Ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) are essential components

of the
translation machinery and have evolved for transmitting genetic information into proteins. In
addition, non
-
coding RNPs such as small nucleolar RNPs (snoRNPs), the RNase P complex
or 7SL or 7Sk
-
contisning

consisting

RNPs have highly specific regula
tory functions within a
cell. The composition as well as the function of such RNPs will be discussed in this chapter.

Like non
-
coding RNAs, mRNAs are also incorporated into mRNA protein complexes and
consistently termed mRNPs. Extensive research during the

last decade has revealed that
mRNAs are packaged into dense protein structures and it is becoming more and more
apparent that highly ordered remodeling of mRNPs is the driving force for mRNA maturation
and subsequent gene expression.
Structure of these RN
As have been shown in
PMID:
19840948

Can you add a connecting sentence: here we show/discuss that….

Ribonuclease P (RNase P)

tRNA genes are transcribed by RNA polymerase III to pre
-
tRNA, which are further matured
to produce functional tRNAs. During tRNA ma
turation, several processing steps are
necessary including chemical modifications of several nucleotides within the tRNA and
removal of sequences both from the 5’ (5’ leader sequence) and 3’ end. RNAse P is and
endonuclease that is essential for the matura
tion of functional tRNAs and mediates processing
of the 5’ leader sequence of precursor tRNAs
[3]
. In bacteria, the RNAse P R
NP consists of
one RNA (RNAse P RNA) of 350


400 nucleotides in length and one single protein of about
14kd
[4]
.
In eukaryotes, however, RNAse P is a large
protein complex with 9 to 10 protein
components associated with one RNAse P RNA
[5]
.
The
RNase P is one of the first
discovered ribozymes i.e. a catalytically active non
-
coding RNA. The associated proteins
within the RNase P RNP support the function of the RNase P RNA. In addition to its role for
tRNA maturation, RNAse P was recently found to
be important for transcription of several
non
-
coding RNAs including tRNAs, 5S rRNA, 7SL RNA and U6 snRNA (see below and
Figure 1)
[6]
.


Small nucleolar RNAs (snoRNAs)

SnoRNAs are 60

300 nucleotides long non
-
coding RNAs that localize to the nucleolus of
eukaryotic cells. snoRNAs associate with specific proteins to form sn
oRNPs, which are
essential for the biogenesis of a number of non
-
coding RNAs including snRNAs, tRNAs and
rRNAs. SnoRNAs mainly guide chemical modifications of specific nucleotides within target
RNAs. SnoRNAs can be classified in two main subgroups, box H/A
CA snoRNAs and the box
C/D snoRNAs. Both subtypes have distinct secondary structures as well as functions
[7]
.

Box H/
ACA snoRNAs fold into a secondary structure of two stem loops with the two stems
connected with each other. Bulges within the two stems are essential for recognition of
complementary target RNA molecules. Box H/ACA snoRNAs interact with the proteins
DKC1 (
dyskeratin), GAR1, NHP2, NOP10 and use base complementartity to guide site
-
specific pseudouridylations to their target RNAs. In addition to the regulation of small RNA
function, snoRNAs are also involved in the action of the well known telomerase holoenzym
e,
which forms an RNP as well. The telomeric RNA (TER) is similar to a H/ACA box snoRNA
and the telomerase holoenzyme represents therefore a H/ACA box
[7]

something appears to
be missing here: a H/ACA
. Box snoRNP?

Box C/D snoRNAs form a large loop, necessary for
target RNA recognition, followed by a short stem which is generated by complementary 3’
and 5’ ends. Box C/D s
noRNAs interact with fibrillarin, NOP 56, NOP58 and NHP2L2
proteins to guide methylation of the 2’ postion of the ribose 2’O
-
ribose (often referred to as
2’
-
O
-
methylation)
[8]
.

Despite their function in rRNA biogenesis a
subclass of snoRNPs, the so
-
called cajal body
-
specific RNPs (scaRNPs) are important for the modification of snRNAs (see below) and are
thus important regulators of the biogenesis of the splicing machinery
[8]
.
Recent find
ings give
rise to another new functionality of snoRNAs. Some snoRNAs are processed to microRNAs
(see below), which in turn function in gene silencing
[9]
.
This sounds like that processed
snoRNA (or dsRNAs, see Mattic RNA paper) have function in gene silencing, which is not
clear.


Small regulatory RNAs

Small regulatory RNAs are characterized by their specific length o
f about 18
-
35 nucleotides
and function in posttranscriptional gene regulation, heterochromatin formation or transposon
silencing. The most common small regulatory RNAs are siRNAs, piwi interacting RNAs
(piRNAs) and miRNAs. Such small RNAs associate with me
mbers of the Argonaute protein
family to form RNPs. Depending on the small RNA component, these RNPs are referred to as
RNA
-
induced silencing complex (RISC, siRNA or miRNAs) or miRNPs (miRNA). Ago
proteins can be phylogenetically divided into two subfamili
es: the Argonaute (Ago) and the
Piwi subfamily. The number of Argonaute proteins is highly variable among species and
ranges from one in S.pombe to 27 in C.elegans. Expression studies in humans revealed that
only the Ago subfamily (human Ago 1
-
4) is ubiqui
tous expressed whereas expression of the
Piwi subfamily (Hiwi 1
-
3, Hili), which binds piRNAs, seems to be restricted to the germ line
[10]
;
[11]
.


Short interfering RNAs (siRNAs)

siRNAs are small RNAs with a length of about 21
-
23 nucleotides. They are processed from
long double stranded RNA precursors by the RNase III enzyme Dicer yielding short double
stranded inte
rmediate RNAs with two nucleotides 3’ overhangs, which is highly characteristic
for RNase III processing. This double stranded intermediate form is subsequently unwound
and only one strand, often referred to as the guide or antisense strand (the other stra
nd is
termed the passenger or sense strand) is incorporated into RISC. SiRNAs guide RISC to
perfectly complementary target RNA molecules and the Argonaute protein within RISC
cleaves the target RNA endonucleolytically. However, not all Argonaute proteins p
osses
endonucleolytic activity. In mammals, for example, only Ago2 cleaves complementary target
RNA molecules and is often referred to as Slicer
[12]
;
[13]
. The process of endon
ucleolytic
cleavage guided by small RNAs is termed RNA interference or short RNAi
[12]
. RNAi was
first discovered in plants where the overexpression of a pigment gene suppressed not only the
expression of the transgene but also the endogenous gene
[14]
. Later on, mechanistic details
of RNAi were identified in Caenorhabditis elegans by Fire and Mello who were awarded the
Nobel Prize in 2006 for their discoveries
[15]
. In some organisms such as Drosophila
melanogaster, endogenously expressed siRNAs are believed to provide innate defen
se
mechanisms against exogenous double stranded RNAs such as viral RNAs. Endogenous
siRNAs have also been found in mammals and they mainly derive from transposon
transcripts, long stem loop structures or double stranded sense
-
antisens transcripts. The exac
t
function of such endo
-
siRNAs is not fully understood
[16]
. RNAi is now widely used as
research tool and may also lead to highly potent and novel way
s for therapy of human
diseases.

In contrast to their well
-
known function in RNAi, siRNAs are also linked to heterochromatin
formation in the fission yeast Schizosaccharomyces pombe. Such siRNAs originate from
repetitive centromeric elements and are theref
ore referred to as repeat
-
associated siRNAs
(rasiRNAs). These small RNAs form a RNP complex together with the sole S.pombe
Argonaute protein, Dicer and a number of other factors the so
-
called RITS (RNA
-
induced
transcriptional silencing) complex
[17]
.


MicroRNAs (miRNAs)

miRNAs are small ~ 22nt long RNA molecules that are produced from endogenous
transcripts. miRNA genes are ex
pressed as long primary miRNA transcripts (pri
-
miRNAs)
that are processed to stem
-
loop
-
structured miRNA precursors (pre
-
miRNAs) by the RNase III
enzyme Drosha, which is part of the nuclear microprocessor complex. Some miRNAs from
form

complete introns with
in pre
-
mRNAs. Interestingly, such miRNAs do not require
cleavage by Drosha but are spliced by the spliceosome giving rise to miRNA precursors. Such
RNA molecules are termed mirtrons
[18]
;
[19]
. Pre
-
miRNAs are exported to the cytoplasm
where they a
re further processed by Dicer. Similar to siRNAs, Dicer produces a short double
stranded RNA intermediate that is further unwound and one strand gives rise to the mature
miRNA, which is incorporated into a RNP often referred to as miRNP or RISC. The other
strand, termed miRNA*, is removed from the cell by degradation. MiRNAs were first
identified in C. elegans where they target the 3’ untranslated region (UTR) of distinct mRNAs
[20]
;
[21]
. In contrast to siRNAs, miRNAs bind to imperfectly complementary binding sites
on their targets leading to inhibition of translation and/or mRNA degradation induced by
poly(A)
-
tail

shortening (deadenylation) and decapping
[22]
. In humans, more than 500
miRNA genes have been identified so far. It is further believed that individual m
iRNAs can
target a large number of mRNAs leading to the conclusion that a large portion of the human
mRNAs is under miRNA control.


Piwi interacting RNAs (piRNAs)

piRNAs are germ line specific small RNAs about 24 to 34 nucleotides in length, which form
piR
NPs with the Piwi subfamily of Argonaute proteins. In contrast to siRNAs and miRNAs,
piRNAs are most likely processed by a dicer independent mechanism. However, the detailed
mode of action is still unclear. PiRNAs derive from large piRNA clusters, transpos
ons or
intergenic repetitive elements and are processed from single stranded RNA
[16]
;
[23]
. PiRNAs
were first discovered in D. melanogaster and linked to repetitive elements like
retrotransposons and it has been demonstrated that they are involved in transposon silencing.
Consisten
tly, piRNAs have been named rasiRNAs in Drosophila. In mice, piRNPs are
important for germ line development as well as spermatogenesis. Similar to Drosophila,
mammalian piRNAs are required for silencing of retrotransposons and other genetic elements
in ger
m line cells, thereby protecting the germ line from severe damage caused by random
insertions of mobile genetic elements
[24]
.


7SL RNA

The non
-
coding 7SL RNA is the core component of the signal recognition particle (SRP). The
SRP RNP is essential for the maturation of secreted or membrane bound proteins. The

SRP
binds the n
N
-
terminal signal peptides that emerge from the ribosome an guides the transport
of the nascent proteins to the endoplasmic reticulum (ER) for further processing and cellular
protein sorting
[25]
. The Mammalian SRP RNP consists of one 7SL RNA molecule with a
size of about 300 nucleotides, which fold into a Y
-
shaped double stranded secondary
structur
e, and six protein subunits (SRP4, SRP19, SRP68, SRP72 SRP14 and SRP9). The
SRP consist of two functional domains: the S
-
domain and the Alu domain. The Alu
-
domain,
which binds to SRP9 and SRP14, mediates a stop of the polypeptide chain elongation until
tra
nslocation of the nascent protein


ribosome complex to the ER membrane. The S
-
domain
binds to SRP19, SRP54, SRP68 and SRP72 and is important for the binding to the signal
peptide sequence of the nascent protein and also for the binding to the ER membrane
[26]
;
[25]
.


7SK RNA

The 7SK RNA is an abundant small RNA with a length of 330 nucleotides. It forms a 7SK
RNP together with the protein kinase CDK9, cyclin T1 (or T2 or K), Hexim1 or Hexim2 and
a number of other proteins. CDK9 together w
ith one of above
-
mentioned cyclins is known as
transcription elngation factor b (p
-
TEFb), which stimulates RNA polymerase II transcription
elongation. The predicted secondary structure of the 7SK RNA is characterized by two stem
loops, which are located at

the 5’ and the 3’ ends. Whereas the 3’ stem loop is important for
7SK RNA stability, the 5’ stem loop of the 7SK RNA is necessary for the interaction with
Hexim1 (hexamethylene bis
-
acetamide
-
inducible mRNA1) or HEXIM2. Both Hexim proteins
inhibit p
-
TEFb k
inase activity, which is necessary for phosphorylation of RNA polymerase II
and transcription elongation
[27]
;
[28]
. Taken together, the 7SK RNA is an important
regulator of RNA polymerase II
-
mediated transcription.


U
-
rich small nuclear RNAs (U snRNAs)

U snRNAs are the core components of the splicing machinery. Like many other non
-
coding
RNAs, U snRNAs are ch
aracterized by extensive secondary structures. All U snRNAs with
the exception of U6 contain a so
-
called Sm binding site that serves as a binding platform Sm
proteins. The Sm proteins SmB, D1, D2, D3, E, F, G from a heptameric ring that interacts
with the
U snRNA. The U6 snRNA associates with like
-
Sm proteins termed LSm2
-
8. Beside
the core Sm proteins, all U snRNAs bind to a number of snRNP
-
specific proteins. During the
splicing reaction, all U snRNPs assemble in a highly order manner to the spliceosome, wh
ich
finally leads to the removal of the intron from the pre
-
mRNA
[29]
.
Since the splicing
machine
ry as well as the molecular mechanisms of splicing will be discussed in detail in the
following chapters, it will not be further discussed here.

Can you add a positive conclusion: The ribonucleoproteins formed by small RNAs have
diverse function in the cel
l and some of them were recruited to form the backbone of the
spliceosome, which is discussed in the next chapter 5.


Table 1: Overview of different cellular small RNPs

There is a figure from chapter 1 that contains more small RNAs, can you incorporate them
into the table?

RNP

Small RNA

function

Ref.

snRNPs

snRNA

Splicing, major components of the
spliceosom
e

[29]

scaRNPs

scaRNA

snRNA modification

[8]

snoRNPs

sn
oRNA

rRNA processing, pre
-
polysomes

[8]

telomerase

snoRNA, TER

telomere length regulation

[7]

miRNP/

RISC

miRNA

Sequence
-
specific gene silencing

[18]

miRNP/

RISC

snoRNA derived
miRNAs

Sequence
-
specific gene silencing

[9]

RISC

siRNA

RNA interference

[12]

piRNP

piRNA

gene silencing gametogenesis

transposon silencig


[16]
;
[24]

RITS

siRNA
(rasiRNAs)

he
terochromatin formation

[17]

RNase P

RNase P RNA

t
-
RNA maturation, transcription of
tRNAs, rRNAs, snRNAs

[5]
;
[4]
;
[6]

SRP

7SL RNA

transport of nascent proteins to the
E
R for further processing

[26]
;
[25]

7SK RNP

7SK RNA

Regulation of transcription
elongation

[27]
;
[28]


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Figure legend

Figure1: Simplified representation of RNP
s and their cellular functions. Small RNA protein
complexes play important roles in mRNA maturation and the regulation of gene expression.
In S.pombe, the RITS complex regulates centromeric heterochromatin formation. Small
RNAs like the 7SK RNA binds and r
egulates transcription elongation factor p
-
TEFb and thus
transcription elongation. U snRNPs are the major components of the spliceosome with U
-
rich
small nuclear RNAs (snRNAs) as RNA components. In the cytoplasm, mRNAs associate with
ribosomal subunits to
initiate the translation process. During translation, the signal recognition
particle (SRP) RNP with 7SL RNA as core RNA component directs signal peptide
-
containing
nascent proteins together with associated ribosomes to the ER for further processing and
ce
llular protein sorting. MiRNPs or RISCs are important regulators of gene expression and
guide post
-
transcriptional gene silencing processes. RNPs play further important roles for the
processing or maturation of RNAs like pre
-
tRNAs, which are processed by R
Nase P. Small
nucleolar RNPs (snoRNAs) or small cajal body RNPs (scaRNAs) are essential for the
maturation of ribosomal RNAs or snRNAs. The well
-
known telomerase enzyme belongs to
the snoRNPs and it restores the length of the telomeric ends of the chromoso
mes, which are
reduced during each cell division cycle. For further details please see the main body of the
text.